U.S. patent application number 13/057161 was filed with the patent office on 2011-06-16 for method of evaluating oral cancer risk in human.
This patent application is currently assigned to INSTITUT CLINIDENT. Invention is credited to Franck Chaubron.
Application Number | 20110143962 13/057161 |
Document ID | / |
Family ID | 41129341 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143962 |
Kind Code |
A1 |
Chaubron; Franck |
June 16, 2011 |
METHOD OF EVALUATING ORAL CANCER RISK IN HUMAN
Abstract
A method of providing a risk evaluation and diagnosis of human
oral cancer, by examining at the presence in human saliva sample of
a combination of particulate nucleic acids from bacteria, virus, as
well as human, and/or the presence of particulate biochemical
volatile organic compounds, which are indicative of an increased
risk of oral cancer.
Inventors: |
Chaubron; Franck; (Venelles,
FR) |
Assignee: |
INSTITUT CLINIDENT
Saint Beauzire
FR
|
Family ID: |
41129341 |
Appl. No.: |
13/057161 |
Filed: |
August 3, 2009 |
PCT Filed: |
August 3, 2009 |
PCT NO: |
PCT/EP09/60050 |
371 Date: |
February 2, 2011 |
Current U.S.
Class: |
506/16 ;
435/287.2; 435/6.11; 435/6.12; 436/64 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/708 20130101; C12Q 1/689 20130101; G01N 33/57407 20130101;
C12Q 1/6886 20130101 |
Class at
Publication: |
506/16 ;
435/6.12; 436/64; 435/6.11; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/48 20060101 G01N033/48; C12M 1/34 20060101
C12M001/34; C40B 40/06 20060101 C40B040/06 |
Claims
1-27. (canceled)
28. An in vitro method of diagnosing a predisposition to oral
cancer in a human subject or of diagnosing an oral cancer in a
human subject, the method comprising collecting and stabilizing a
crude saliva sample from said human subject, and performing at
least one of the following steps a) and/or b): a) Analyzing a fluid
fraction of said stabilized saliva sample by detecting specific DNA
or RNA sequences of human, bacterial or viral origin in said
fluidic fraction, b) Analyzing the volatile fraction extracted from
said stabilized saliva sample by detecting in said volatile
fraction at least one biochemical organic compound; wherein the
detection of at least one DNA or RNA sequence as defined in a)
and/or at least one biochemical organic compound as defined in b),
is indicative of a risk or a predisposition to develop oral
cancer.
29. The method of claim 28, wherein said specific DNA or RNA
sequences are chosen among: i) human sequences selected from SSAT
mRNA (SEQ ID No 62), H3F3A mRNA (SEQ ID No 63) and IL8 mRNA (SEQ ID
No 64); and/or ii) sequences of bacteria selected from
Capnocytophaga gingivalis (ATCC 33624), Prevotella melaminogenica
(ATCC 25845), Streptococcus mitis (ATCC 15914) and Micrococcus
luteus (ATCC 53598D); and/or iii) the viral sequences of human
papillomavirus.
30. The method according to claim 29, wherein the detection of at
least two human mRNA sequences as specifically defined in i) and at
least one bacterial sequence as specifically defined in ii) in the
saliva of a human subject indicates that said human subject has a
high risk of developing an oral cancer.
31. The method according to claim 30, wherein the detection of the
human mRNA of H3F3A (SEQ ID No 63), of the human mRNA of SSAT (SEQ
ID No 62) and of the bacterial genome of Streptococcus mitis (ATCC
15914) in the saliva of a human subject indicates that said human
subject has a high of developing an oral cancer.
32. The method according to claim 29, wherein the human
papillomavirus is human papillomavirus 16 (ATCC 45113) or human
papillomavirus 18 (ATCC 45152).
33. The method according to claim 28, wherein the crude saliva is
stabilized by using a solution comprising a salt such as guanidium
thiocyanate, and/or ammonium sulfate, and/or sodium azide, and
optionally exo and/or endonuclease inhibitors.
34. The method according to claim 28, wherein specific DNA or RNA
sequences are detected by incubating said genomic DNA and total RNA
with a thermostable enzyme with RNA-dependent Reverse Transcriptase
activity and with DNA-dependent Polymerase activity.
35. The method according to claim 34, wherein the combination of
RT-PCR and PCR is performed in a single-tube reaction.
36. The method of claim 28, wherein said at least one biochemical
organic compound is selected in the group consisting of:
2,3-pentanedione (CAS number 600-14-6), 3-methyltiophene (CAS
number 616-44-4), acetone (CAS number 67-64-1), hexanenitrile (CAS
number 628-73-9), benzaldehyde (CAS number 100-52-7),
3-methyl-2-pentanone (CAS number 565-61-7), 2,3-butanedione (CAS
number 431-03-8), 2-propanol (CAS number 67-63-0), ethyl acetate
(CAS number 141-78-6), 1-propanol (CAS number 71-23-8), hexanal
(CAS number 66-25-1), 5-methyl-3-hexen-2-one (CAS number
5166-53-0), m-xylene (CAS number 108-38-3), p-xylene (CAS number
106-42-3), 2-methyl-2-butenal (E) (CAS number 497-03-0), phenol
(CAS number 108-95-2), butanal (CAS number 123-72-8),
methylbutanone (CAS number: 563-80-4), 2-methyl-2-butene (CAS
number 513-35-9), 2-methyl-1-propene (CAS number 115-11-7) and
(cis) 1,2 dimethyl-cyclopropane (CAS number: 930-18-7).
37. The method according to claim 36, wherein the detection of at
least one biochemical organic compound chosen among
2,3-pentanedione (CAS number 600-14-6), 3-methyltiophene (CAS
number 616-44-4), acetone (CAS number 67-64-1), hexanenitrile (CAS
number 628-73-9), benzaldehyde (CAS number 100-52-7),
3-methyl-2-pentanone (CAS number 565-61-7), 2,3-butanedione (CAS
number 431-03-8), 2-propanol (CAS number 67-63-0), ethyl acetate
(CAS number 141-78-6), 1-propanol (CAS number 71-23-8), hexanal
(CAS number 66-25-1), 5-methyl-3-hexen-2-one (CAS number
5166-53-0), m-xylene (CAS number 108-38-3), p-xylene (CAS number
106-42-3), 2-methyl-2-butenal (E) (CAS number 497-03-0) in the
volatile fraction of saliva of an human subject indicates that said
human subject has a high risk of developing an oral cancer.
38. The method according to claim 36, wherein the detection of the
biochemical organic compounds of the group comprising:
hexanenitrile, 2,3-pentanedione, 3-methylthiophene and acetone in
the volatile fraction of saliva of a human subject indicates that
said human subject is developing an oral cancer.
39. The method according to claim 28, wherein the detection of the
mRNA sequence of H3F3A (SEQ ID No 63), of SSAT (SEQ ID No 62) and
the detection of the biochemical organic compounds hexanenitrile,
2,3-pentanedione, 3-methylthiophene and acetone in the saliva of a
human subject indicates that said human subject is developing an
oral cancer.
40. The method according to claim 28, wherein biochemical organic
compounds are detected when the expression level of said compound
is at least superior to 1.5 fold the mean expression level of said
compound in the normal population.
41. The method according to claim 36, wherein the detection of at
least one biochemical organic compound chosen in the group
comprising: 2-methyl-2-butene (CAS number 513-35-9),
2-methyl-1-propene (CAS number 115-11-7) and (cis) 1,2-dimethyl
cyclopropane (CAS number 930-18-7) in the volatile fraction of
saliva of a human subject indicates that said human subject is not
developing an oral cancer.
42. The method according to claim 28, wherein the volatile fraction
of saliva is extracted from crude saliva sample by heating said
saliva sample for at least 10 minutes at 40.degree. C. by using a
Solid-phase Microextraction (SPME) with a CAR/PDMS fiber.
43. The method according to claim 28, wherein the biochemical
organic compounds in said volatile fraction are detected by using a
chromatograph in gas phase coupled to a mass spectrometer.
44. The method according to claim 28, wherein specific DNA or RNA
sequences are detected when the expression level of said sequences
is equivalent or superior to 2 fold the mean expression level of
said sequences in the normal population.
45. The method according to claim 28, comprising amplifying and
detecting at least one DNA or RNA sequence chosen in the group
comprising: SEQ ID NO 62 to 70.
46. The method according to claim 28, comprising the steps of: a)
collecting a sample of crude saliva of said human subject in a
sterile device, b) stabilizing said sample by adding a solution
comprising a guanidium salt, such as guanidinum thiocyanate, and/or
ammonium sulfate, and/or sodium azide, and optionally exo and/or
endonuclease inhibitors, c) extracting total nucleic acid of
bacteria, virus, and human origins from the previously obtained
stabilized saliva sample, d) precipitation and purification of
total nucleic acids, e) incubating the purified total nucleic acid
with a thermostable enzyme with RNA-dependant reverse transcriptase
activity and with DNA-dependant polymerase activity and
polynucleotide primers under conditions which allow the reverse
transcriptase activity of said thermostable enzyme to synthetize
cDNA from the ribonucleic and amplification of genomic DNA and cDNA
at a detectable level by Polymerase Chain Reaction, f) detecting in
an assay the amplified DNAs sequences by hybridization with one or
more polynucleotide probes specific to: i) human sequences selected
from SSAT mRNA (SEQ ID No 62), H3F3A mRNA (SEQ ID No 63) and IL8
mRNA (SEQ ID No 64); or ii) sequences of bacteria selected from
Capnocytophaga gingivalis (ATCC 33624, SEQ ID No 65), Prevotella
melaminogenica (ATCC 25845, SEQ ID No 66), Streptococcus mitis
(ATCC 15914, SEQ ID No 67) and Micrococcus luteus (ATCC 53598D, SEQ
ID No 68), or iii) sequences of virus selected from human
papillomavirus 16 (ATCC 45113, SEQ ID No 69) and human
papillomavirus 18 (ATCC 45152, SEQ ID No 70).
47. The method according to claim 46, wherein primers and probes
used to amplify and detect at least one sequence of i), ii) or iii)
are selected from the group consisting of SEQ ID No 1 to SEQ ID No
60.
48. A kit to practice the method according to claim 46, comprising
primers and probes sequences to amplify and detect at least one
sequence of i), ii) or iii), including at least one of the
following: a) a sterile device to collect a saliva sample,
optionally including a control nucleic acid, and a collecting
reagent, b) a spray dry preservative reagent, c) a resin having
affinity for total nucleic acid, d) a thermostable enzyme with
RNA-dependant reverse transcriptase activity and with DNA-dependant
polymerase activity and polynucleotide.
49. A kit according to claim 46, wherein primers are selected from
the group consisting of: SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ
ID No 8, SEQ ID No 10, SEQ ID No 12, SEQ ID No 14, SEQ ID No 16,
SEQ ID No 18, SEQ ID No 20, SEQ ID No 21, SEQ ID No 23, SEQ ID No
25, SEQ ID No 27, SEQ ID No 29 and SEQ ID No 31.
50. A microarray for diagnosing a predisposition to oral cancer in
a human subject comprising probes sequences selected from the group
consisting of SEQ ID No 32 to SEQ ID No 60.
Description
[0001] The present application claims the priority of the US
provisional patent application filed on Aug. 4, 2008, under the
Ser. No. 61/086,019.
[0002] The present invention relates to a method of providing a
risk evaluation and diagnosis of human oral cancer by examining, in
a saliva sample of a human subject, the presence of particular
nucleic acids of bacteria, virus, and/or human origin, as well as
volatile biochemical organic compounds, a combination of which
being indicative of an increased risk of oral cancer.
BACKGROUND OF THE INVENTION
[0003] Cancers of the oral cavity accounted for 274,000 cases in
2002, with almost two-thirds of them in men. The world area with
the highest incidence is Melanesia (31.5 per 100,000 in men and
20.2 per 100,000 in women). Rates in men are high in western Europe
(11.3 per 100,000), southern Europe (9.2 per 100,000), south Asia
(12.7 per 100,000), southern Africa (11.1 per 100,000), and
Australia/New Zealand (10.2 per 100,000). In females, incidence of
oral cancer is relatively high in southern Asia (8.3 per 100,000).
These patterns reflect prevalence of specific risk factors, such as
tobacco/alcohol, lack of dental and oral health and the chewing of
betel quid in south central Asia and Melanesia. Moreover, for oral
cavity cancer, the overall 5-year survival rates have not improved
in the past several decades, remaining low at approximately 30-50%
(Epstein, J. B. et al. 2002 J Can Dent Assoc 68: 617-621; Mao, L.
et al. 2004 Cancer Cell 5: 311-316).
[0004] In addition, most of oral cancers are initially asymptomatic
and are not diagnosed or treated until they reach an advanced
stage. As of today, patients are questioned about associated risk
to oral cancer (smoker, alcohol) followed by clinical inspection of
oral cavity. Nonetheless, as indicated above early stages such as
preneoplastic states and states of early tumor recurrence show no
tissue damages that are visible by dentists or physicians.
[0005] Thus, there is a need for a method of assessing risk factor,
for early diagnosis, and improved prognosis. In this regard, one
problem is to provide a method that can be performed routinely and
in the usual practice or laboratories.
[0006] The present invention disclosed a reliable and sensitive
diagnostic method applied to the saliva of human subjects.
[0007] Saliva is a clear, slightly acidic fluid that contains a
number of inorganic and organic constituents important to oral
health. Whole saliva is a mix of secretions from major and minor
salivary glands and gingival crevicular fluid, which contains
sloughed host cells, bacteria and food debris. Therefore, saliva is
not a passive "ultrafiltrate" of serum, but contains a distinctive
composition of enzymes, hormones, antibodies, and other molecules
(Rehak, N. N. et al. 2000 Clin Chem Lab Med 38:335-343; Wong D T,
American Scientist, vol 96, 2008). For example, saliva contains a
large number of proteins that aid in the protection of oral cavity
tissues, including mucins, amylases, agglutinins, lisozymes,
peroxidases, lactoferrin and secretory IgA. Whole saliva contains
normal epithelial cells and leukocytes that can be pelleted, and
from which one can easily recover genomic DNA and mRNA, potentially
used to find genomic markers of several diseases. Indeed, most of
the DNA or RNA extracted from crude saliva was found to be of viral
or bacterial origin (Stamey, F. R. et al. 2003 J Virol Methods
108:189-193; Mercer, D. K. et al. 2001 FEMS Microbiol Lett
200:163-167) and of human extra or intracellular origin. Also, many
groups have focused their study and diagnostic tests on the
supernatant and thus cell-free phase of whole saliva, which
contains many analytes such as free mRNA (Zimmermann B G et al,
Oral Oncology 2008, 44, 425-429).
[0008] In the past 10 years, the use of saliva as been successfully
applied in diagnostics (Streckfus, CF. & Bigler, L. R. 2002
Oral Dis 8:69-76). Diagnostic biomarkers in saliva have been
identified for monitoring caries, periodontitis, salivary gland
diseases, and systemic disorders, e.g., hepatitis and HIV (Lawrence
H. P. et al, 2002 J Can Dent Assoc 68: 170-174). Also, oral
bacteria have been reported to be elevated in oral and esophageal
cancer lesions (Mager D. L. et al. J Transl Med. 2005; 3: 27,
Hooper J. S et al., Journal of Clinical Microbiology, May 2006, P
1719-1725). The reason for these shifts in bacterial colonization
of cancer lesions is unclear. Mechanistic studies of bacterial
attachment provide some insights and research has repeatedly shown
that oral bacteria demonstrate specific tropisms toward different
biological surfaces in the oral cavity such as the teeth, mucosa,
and other bacteria. There is less time in oral cavity, for a
complex biofilm to develop on soft tissue surfaces; thus, a premium
is placed on potent mechanisms of adhesion. The differences in
bacterial tropisms for specific oral sites suggest that different
intra-oral surfaces and bacterial species have different receptors
and adhesion molecules that dictate the colonization of different
oral surfaces. Certain glycoconjugates serve as receptors for
specific bacteria and recent reports support the notion that shifts
in the colonization of different cancer cells are associated with
observed changes in cell surface receptors. Hence, Mager D. L. et
al showed that the salivary microbiota in subjects with an oral
squamous cell carcinoma (OSCC) lesion differs from that found in
OSCC-free controls. Bacterial counts were determined for each
species, averaged across subjects in the 2 subject groups, and
significance of differences between groups determined using the
Mann-Whitney test and adjusted for multiple comparisons;
interestingly, it appeared that the bacteria strains Capnocytophaga
gingivalis, Prevotella melaminogenica, Streptococcus mitis and
Micrococcus luteus were particularly present in patients having
OSCC and were therefore suggested to serve as diagnostic markers
for oral cancer. However, as it is demonstrated in (ref), these
particulate bacteria strains were poorly associated with oral
cancer (a maximal sensitivity of 80%) (Mager D. L. et al). Also, it
has been shown in Li et al (Journal of Applied Microbiology, 2004,
97, 1311-1318) that the presence in saliva of significant high
numbers of specific alive bacteria (40 different strains have been
identified in this study and more than 200 specific alive bacteria
have been described in the oral cavity), could be associated to the
biofilm formation, colonization of the oral cavity and lack of oral
hygiene that are often associated to oral cancer development in
developing countries. However, one can not predict from Li et al
that the particulate strains Capnocytophaga gingivalis, Prevotella
melaminogenica, Streptococcus mitis and Micrococcus luteus can
serve as reliable diagnostic markers for human oral cancer. This is
the reason why, to date, no reliable and very sensitive
bacteria-based diagnostic test has ever been proposed to diagnose
oral cancer in saliva.
[0009] It is interesting to note that the majority of species
isolated were saccharolytic and acid tolerant and are known to
produce short-chain organic acids from carbohydrates and
consequently to lower the pH of their local environment. Raghunad
N. et al. (J. Radiol. 2003; 76. S11-S22) described the
microenvironment of solid tumors as is typically hypoxic, with an
acidic extracellular pH so it is not surprising that there might be
a degree of selectivity in favor of acid tolerance. Other factor
could be production of DNA oxidative damage generated by oral
bacteria. Takeuchi T. et al. (2000, FEMS Microbiol Lett. November
1; 192(1):133-8), investigated the mechanism of the oxidative DNA
damage induction by exposure to O.sub.2 in Prevotella
melaminogenica, a strict anaerobe found in oral cavity and some
dental diseases. Results indicate that in Prevotella
melaminogenica, exposure to O.sub.2 generated and accumulated
O.sub.2 and H.sub.2O.sub.2, and that a crypto-OH radical generated
through H.sub.2O.sub.2 was the active species in the 8OHdG
induction, highly responsible for oxidative DNA damage in human
cells (genotoxic effect), possible cause of mammalian cell
tumorigenicity in the oral environment. Other bacterial pathogens
have been described as possible source of oral tissus
cancerisation.
[0010] James J. Closmann, also found that oral and oropharyngeal
squamous cell carcinoma (OOSCC) have been linked to high-risk HPV
strains, the same strains that cause cervical cancer in women.
D'Souza et al. (N Engl J. Med. 2007 May 10; 356(19):1944-56)
concluded that oral HPV infection is strongly associated with
oropharyngeal cancer among subjects with or without the established
risk factors of tobacco and alcohol use. The degree of association
increased with the number of vaginal-sex and oral-sex partners.
Oropharyngeal cancer was associated with oral HPV type 16 (HPV-16)
infection; oral infection with any of 37 types of HPV and
seropositivity for the HPV-16 L1 capsid protein. HPV-16 DNA was
detected in 72% of 100 paraffin-embedded tumor specimens, and 64%
of patients with cancer were seropositive for the HPV-16
oncoprotein E6, E7, or both. HPV-16 L1 seropositivity was highly
associated with oropharyngeal cancer among subjects with a history
of heavy tobacco and alcohol use. The association was similarly
increased among subjects with oral HPV-16 infection, regardless of
their tobacco and alcohol use. Herrero R et al. (J Natl Cancer
Inst. 2003 Dec. 3; 95(23):1772-83) conducted a multicenter
case-control study of cancer of the oral cavity and oropharynx in
nine countries with 1670 case patients (1415 with cancer of the
oral cavity and 255 with cancer of the oropharynx) and 1732 control
subjects HPV DNA was detected by polymerase chain reaction (PCR).
Antibodies against HPV16 L1, E6, and E7 proteins in plasma were
detected with enzyme-linked immunosorbent assays. Multivariable
models were used for case-control and case-case comparisons. HPV
DNA was detected in biopsy specimens of 3.9% of 766 cancers of the
oral cavity with valid PCR results and 18.3% of 142 cancers of the
oropharynx with valid PCR results. HPV16 DNA was found in 94.7% of
HPV DNA-positive case patients. Co Consequently, it was suggested
that antibodies against HPV16 L1 were associated with a high risk
for cancers of the oral cavity and the oropharynx. Antibodies
against HPV16 E6 or E7 were also associated with risk for cancers
of the oral cavity and the oropharynx. HPV virus is even more of an
indicator that patients should visit the dentist twice a year to
identify anomalies early. Moreover, Rosenquist K. et al. (Acta
Otolaryngol. 2007 September; 127(9):980-7), described that
high-risk orally HPV-infected patients have a significantly higher
risk of recurrence/second primary tumors compared with high-risk
HPV-negative patients. Therefore, diagnosis of HPV in saliva of
human subjects appears to be linked to a higher risk of developing
oral cancer but also for monitoring associated treatment
(antivirus) effects. However, to date, no HPV-based diagnostic test
has ever been proposed to diagnose oral cancer in saliva.
[0011] On another hand, genetic aberrations of cancer cell lead to
altered gene expression patterns (mRNA expression), which can be
identified long before the resulting cancer phenotypes are
manifested. Changes that arise exclusively or preferentially in
cancer, compared with normal tissue of same origin, can be used as
molecular biomarkers (Sidransky, D. 2002 Nat Rev Cancer 2:210-219,
2002). Accurately identified, biomarkers may provide new avenues
and constitute major targets for cancer early detection and cancer
risk assessment. A variety of nucleic acid-based biomarkers have
been demonstrated as novel and powerful tools for the detection of
cancers (Hollstein, M. et al. 1991 Science 253:49-53; Liu, T. et
al. 2000 Genes Chromosomes Cancer 27:17-25; Groden, J. et al. 1991
Cell 66:589-600). These nucleic-acid based biomarkers are mostly
studied in the cell-free phase of whole saliva samples, i.e. in
filtrated or centrifugated saliva samples (Zimmermann B G et al,
Oral Oncology 2008, 44, 425-429). However, there are only a limited
number of reports demonstrating tumor cell DNA heterogeneity in
saliva of oral cancer patients (Liao P. H. et al. 2000 Oral Oncol
36:272-276; El-Naggar, A. K. et al. 2001 JMolDiagn 3:164-170).
Serum circulating human mRNA have been described for oral cancer
detection (Yang Li et al, 2006, Journal of Clinical Oncology vol 24
number 11, 1754-1760). Using microarrays, profiling human salivary
transcriptome (mRNA) have been realized on healthy controls and
cancer patients (Hu S, and al. 2006, J Dent Res. December;
85(12):1129-33). The different gene expression patterns were
analyzed by combining a T test comparison and a fold-change
analysis on 10 matched cancer patients and controls. The predictive
power of these salivary mRNA biomarkers was analyzed by receiver
operating characteristic curve and classification models. This
microarray analysis showed that there are 1,679 genes exhibiting
significantly different expression level in saliva between cancer
patients and controls, among which 7 cancer-related the mRNA of
IL8, IL1B, DUSP1, HA3, OAZ1, S100P, SAT are found. Combinations of
these biomarkers yielded sensitivity (91%) and specificity (91%) in
distinguishing oral cancer from the controls (WO2006/020005 or
WO2005/081867).
[0012] Saliva is a mixture of secretions from multiple salivary
glands, including the parotid, submandibular, sublingual and other
minor glands lying beneath the oral mucosa. As mentioned before,
human saliva harbors a wide spectrum of peptides and proteins that
constitutes the human salivary proteome. What has been less studied
is the presence of organic biochemical compounds in the saliva.
[0013] Biochemical organic compounds can be enzymes, hormones,
inorganic ions, peptides, proteins, carbohydrates, vitamins,
lipids, fatty acids and volatile compounds. They can be measured by
many techniques and devices, either optical technologies (for
example laser absorption spectroscopy, mid infra red absorption
spectroscopy, laser magnetic resonance spectroscopy, proton
transfer reaction mass spectrometry . . . ) or non-optical
technologies (gas chromatography, mass spectrometry, etc. . . . )
(Mashir A, Advanced Powder Technology, 2009).
[0014] Only one study has ever compared the biochemical organic
content of a fraction of saliva from healthy or sick-patients
(Volozhin et al. Stomatologiia (mosk), 2001; 80(1):9-12). In this
study, patients with chronic generalized periodontitis and patients
with chronic generalized gingivitis and periodontitis have been
tested with air from the oral cavity and liquid samples were
collected by washing the oral cavity with sterile water. Chemical
compounds of the air and the washed liquid were analyzed by
chromato-mass-spectrometry, gas-adsorption and gas-liquid
chromatography. The content of dimethyl sulphide, dimethyl
disulphide increased in the oral air and such volatile short chain
fatty acids (VSCFA) as butyrate, propionate, acetate rose, but
their aldehydes (butyraldehyde, acrolein, acetaldehyde) decreased
in oral fluid during periodontitis. It was also shown that volatile
short-chain fatty acids (propionate, butyrate and acetate) of
bacterial and tissue origin are important factors of pathogenesis
of oral tissue inflammation (Volozhin et al. Stomatologiia (mosk),
2001; 80(1):9-12). In this study, the organic compounds have been
analyzed in air from the oral cavity and rincing liquid collected
by washing the oral cavity with sterile water but not in the
volatile fraction of the raw saliva.
[0015] Contrary to saliva, the presence of volatile organic
molecules in exhaled breath has been well studied and was shown to
contain a lot of biochemical organic compounds: in 1971, using
gas-liquid partition chromatography analysis, Linus Pauling
demonstrated the presence of 250 substances in exhaled breath
(Pauling L. et al. Proc. Natl. Acad. Sci. USA 68 (1971) 2374-2376).
In 1990, the development of very sensitive modern mass spectrometry
(MS) and gas chromatography mass spectrometry (GC-MS) instruments,
gives identity to thousands of unique substances in human exhaled
breath (Mashir A, Advanced Powder Technology, 2009). These
substances include elemental gases like nitric oxide and carbon
monoxide and a multitude of other volatile organic compounds.
Furthermore, exhaled breath also carries aerosolized droplets
collected as exhaled breath condensate that have non-volatile
compounds that can be captured by a variety of methods and analyzed
for a wide range of biomarkers from metabolic end products to
proteins. Breath analysis is now used to diagnose and monitor
asthma, pulmonary hypertension, respiratory diseases,
gastrointestinal diseases, critical illness, to check for
transplant organ rejection, and to detect lung cancer, and breast
cancer (Mashir A, Advanced Powder Technology, 2009; Chan H. P. et
al, Lung Cancer, 2009). However, it is noteworthy that breath
analysis has never been proposed to detect oral cancer in human
subject.
[0016] Interestingly, it appeared that the biochemical organic
molecular composition of saliva has never been compared between
patients suffering from cancer, e.g. oral cancer, and healthy
individuals. Moreover, the biochemical organic molecular
composition of the volatile fraction of saliva has never been
studied so far.
[0017] The volatile fraction corresponds to the evaporated part of
the fluid fraction of saliva. This volatile fraction contains some
organic compounds that are not found in the raw saliva sample
without the evaporation process, even after its filtration.
[0018] Also, it is important to note that the molecular content of
saliva is not comparable at all to the composition of exhaled
breath, which is mostly a reflect of lung molecules (Song G, et al.
Quantitative breath analysis of volatile organic compounds of lung
cancer patients, Lung Cancer (2009)). Hence, the molecular
composition of the volatile fraction of saliva can not be inferred
from the data issued from the breath analysis. To date, no
exhaustive analysis of the biochemical content of the volatile
fraction of saliva has never been disclosed, a fortiori in the
context of oral cancer.
[0019] One of the values of saliva is the ease of sampling and
subject compliance for sample collection, which includes field
applications as well as home collection. However, the study of the
biochemical compounds present in saliva (either in fluid or in the
volatile fraction) for clinical application appeared to be
difficult since it is necessary to stabilize and maintain their
integrity for at least several days at room temperature.
[0020] It has already been shown that the RNAprotect.RTM. Saliva
reagent (RPS, Qiagen Inc, Valencia, Calif.) could stabilize RNA in
samples at room temperature for up to 12 weeks (Park N.J., Clin
Chem 2006; 52:2303-4). Also, Jiang J et al showed that it is
possible to use the RPS for stabilization of DNA and proteins in
saliva only when endogenous cells are previously removed by
centrifugation or by filtration (Jiang J et al, Archives of Oral
Biology, 2008).
[0021] As far as biochemical organic compounds are concerned, they
are degraded by the microflora, food and dental care products at
room temperature. Importantly, nobody has ever proposed a way to
protect these sensitive compounds from the degradation occurring at
room temperature. A fortiori, a buffer enabling the stabilization
of both nucleic acids and biochemical organic compounds for several
days at room temperature has never been described so far.
[0022] Therefore, it is still a major challenge to stabilize all
the components of saliva, especially nucleic acids and organic
compounds, without any filtration or centrifugation steps, for a
long time at room temperature.
[0023] In this context, the present inventors show here for the
first time that: [0024] i) it is possible to stabilize the nucleic
acids and organic compounds present in saliva during at least 10
days in an appropriate buffer, [0025] ii) it is possible to detect
a high risk of developing oral cancer by analyzing the level of
only few particular biomarkers in the fluid fraction of a
stabilized sample of saliva, [0026] iii) it is possible to extract
a volatile fraction from a sample of stabilized saliva, and to
detect therein several organic volatile compounds in a significant
amount, [0027] iv) it is possible to detect a high risk of
developing oral cancer by analyzing the level of only few
particular biochemical compounds in said volatile fraction of
stabilized saliva.
[0028] Importantly, it is to note that no test for predicting
and/or diagnosing oral cancer using the volatile fraction of saliva
has ever been proposed so far.
[0029] More importantly, the particular biochemical compounds
hereafter identified have never been associated with cancer so
far.
[0030] The present invention therefore discloses: [0031] i) a
method for stabilizing crude saliva samples of human subjects,
[0032] ii) a method for detecting a high risk of developing oral
cancer, by analyzing, in a one-step reaction, the level of
particular biomarkers in the fluid fraction of said stabilized
saliva, [0033] iii) a method for extracting the volatile fraction
of stabilized saliva samples, and to analyze its content in
biochemical organic compounds, [0034] iv) a method for detecting a
high risk of developing oral cancer, by analyzing the level of
particular organic biochemical compounds in the volatile fraction
of stabilized saliva.
[0035] Finally, specific combinations of biological parameters
associated with high risk of oral cancer have been identified,
highlighting that it is possible to obtain a reliable and sensitive
prognosis/diagnosis test of oral cancer from a unique sample of
stabilized saliva.
FIGURE DESCRIPTION
[0036] FIG. 1 shows an histogram representing the 2 groups of
saliva samples after a Factorial Discriminant Analysis (FDA) on
ratios data according to the factor <<tumor/healthy>>:
5 ratios allow to separate 98.077% of the samples in the 2 groups
<<tumor>> and <<reference group>> (only 1
reference sample is classified in the group <<tumor>>
and not in the reference group).
DESCRIPTION
[0037] The present invention disclose a new reliable, sensitive and
easy to handle diagnostic test of oral cancer in human subject.
[0038] The present invention relates to an in vitro method of
diagnosing a predisposition to oral cancer in a human subject, the
method comprising stabilizing a crude saliva sample from said human
subject, and performing at least one of the following steps a)
and/or b): [0039] a) Analyzing a fluid fraction of said stabilized
saliva by detecting specific DNA or RNA sequences of human,
bacterial or viral origin in said fluidic fraction, [0040] b)
Analyzing a volatile fraction extracted from said stabilized saliva
by detecting in said volatile fraction at least one biochemical
organic compound; wherein the detection of at least one DNA or RNA
sequence as defined in a) and/or at least one biochemical organic
compound as defined in b), is indicative of a risk or a
predisposition to oral cancer.
[0041] The present invention enables to determine if a human
subject is predisposed of developing an oral cancer or not.
[0042] When a human subject is "diagnosed to have a predisposition
to oral cancer" or is found to be "predisposed to oral cancer", it
means that he has a higher risk of developing an oral cancer than
the mean healthy population in the future. In other words, he is
thought to be predisposed for developping an oral cancer in the
early or far future. In the context of the invention, a human
subject is said to be "predisposed to develop oral cancer" when he
has a risk superior 70%, preferably 80%, more preferably 90% and
even more preferably 95% of developing oral cancer in a short or
far future as compared to a normal healthy population. This cancer
predisposition is generally linked to a genetic cause.
[0043] The present invention also enables to determine that a human
subject is "not predisposed to develop an oral cancer". In this
case, it means that he has a poor risk of developing oral cancer in
the future. For example, it means that he has a risk of developing
an oral cancer in the future lower than 10%, preferably lower than
5% as compared to the normal healthy population. It generally means
that he has at least 90% of chance not to have oncogenic mutations
in his genome.
[0044] The present invention also enables to diagnose an oral
cancer in a human subject. As a matter of fact, the present
invention disclosed an in vitro method of diagnosing an oral cancer
in a human subject, comprising stabilizing a crude saliva sample
from said human subject, and performing at least one of the
following steps a) and/or b): [0045] b) Analyzing a fluid fraction
of said stabilized saliva by detecting specific DNA or RNA
sequences of human, bacterial or viral origin in said fluidic
fraction, [0046] b) Analyzing a volatile fraction extracted from
said stabilized saliva by detecting in said volatile fraction at
least one biochemical organic compound; wherein the detection of at
least one DNA or RNA sequence as defined in a) and/or at least one
biochemical organic compound as defined in b), is indicative of a
risk of developing oral cancer.
[0047] The method of the invention is thus dedicated to estimate a
risk for a human subject of developing an oral cancer. This risk
can be either a high risk of developing an oral cancer or a low
risk of developing an oral cancer.
[0048] As used herein, when a human subject has a risk "of
developing" an oral cancer, it means that he has a risk "to be
developing" an oral cancer at the time of the collection of the
saliva sample.
[0049] In the context of the invention, a human subject is said "to
have a high risk of developing an oral cancer" when he has a risk
at least superior to 60%, preferably to 70%, more preferably to 80%
and even more preferably to 90% of developing an oral cancer. In
other words, the human subject has a much higher probability to
develop an oral cancer as compared to the normal population or to a
human subject in which none of the organic compound is detected. In
the context of the present invention, when a human subject has a
risk superior to 97% to be developing an oral cancer, it is said
that the human subject "is developing an oral cancer".
[0050] This oral cancer can be initiating or well-established. In
one embodiment of the invention, the level of expression of
particulate biochemical organic compounds can potentially indicate
the grade of the oral cancer from which the human object is
suffering.
[0051] The method of the invention also enables to determine if a
human subject has a low risk to be developing an oral cancer. In
the context of the invention, the human subject has a low risk of
developing an oral cancer when he has a risk of developing an oral
cancer lower than 10%, preferably lower than 5% as compared with
the normal population. In other words, the human subject has a
chance superior to 90%, preferably 95% to be healthy, at least as
far as oral cancer is concerned. In the context of the invention,
when a human subject has a risk inferior to 5% of being developing
an oral cancer, it is said that the human subject is not developing
an oral cancer at the time of the collection of the saliva
sample.
[0052] In the context of the invention, the term "oral cancer"
triggers the following cancers: cancer of the oral cavity, cancer
of the oropharynx, oropharyngeal squamous cell carcinoma (OSCC),
head and neck squamous cell carcinoma, preferably oral squamous
cell carcinoma.
[0053] In the context of the invention, "collecting a crude saliva
sample" is obtained by receiving, in a sterile device, a sample of
the saliva that has been spitted by the human subject. A collecting
reagent, for example a citrate buffer, may be added to the sample.
This sample is then treated so as to stabilize it for later
analysis, and to prepare it to nucleic acid and/or volatile
fraction extraction.
[0054] It is one aspect of the present invention to provide a way
to stabilize crude samples of saliva, so as to maintain high
amounts of genetic markers and biochemical components as present in
the initially spitted and collected saliva and to favour nucleic
acid and/or biochemical volatile elements extraction. The present
invention enables to maintain preferably at least 70%, more
preferably 80% and even more preferably 90% of the amount of
nucleic acids and biochemical organic components initially present
in the spitted and collected saliva.
[0055] More precisely, the method of the invention enables to
protect the raw components of the sample from degradation during at
least 10 days at room temperature. This stabilization step is
performed by adding to the crude sample a so-called "saliva
preservation solution" comprising at least a preservation reagent,
which is preferably a buffer comprising a salt capable of a)
opening the membrane of bacteria and human cells, b) blocking
nuclease activity, c) precipitating the nucleic acid and d)
reducing the vapour tension of volatile compounds without allowing
the degradation of said compounds. This salt is preferably a salt
such as guanidinum thiocyanate, and/or ammonium sulfate and/or
sodium azide, preferably sodium azide. This salt is employed
preferably at a concentration range between 20 mM and 6 M, and more
preferably at 40 mM.
[0056] The present invention therefore discloses a method for
stabilizing the raw components of a crude saliva sample (such as
nucleic acid and biochemical organic compounds) during at least 10
days at room temperature, said method comprising adding to the
crude saliva sample a salt such as guanidinum thiocyanate, and/or
ammonium sulfate and/or sodium azide. The salt is preferably sodium
azide and is present at a concentration of about 40 mM.
[0057] Therefore, by "stabilized" saliva sample, it is meant to
refer to samples in which nucleic acid species and organic volatile
compounds have been preserved from degradation caused by the
microflora, food and dental care products, during at least 10 days
at room temperature.
[0058] In a first aspect, the present invention provides a method
of diagnosing a risk or a predisposition to oral cancer in a human
subject, simply by using a stabilized sample of its saliva and
analyzing its fluid fraction by detecting specific DNA or RNA
sequences of human, bacterial or viral origin.
[0059] As used herein, the "fluid fraction" (or "fluidic fraction")
of a saliva sample is the liquid phase of the collected saliva, and
contains cells as well as free nucleic acids and organic
compounds.
[0060] In the method of the invention, and contrary to what has
been described so far, there is no need to remove the cells from
the crude saliva samples to obtain the fluid fraction. Therefore,
the saliva fluid samples are preferably not centrifugated and not
filtrated.
[0061] As used herein, the expression "DNA or RNA sequences" refer
to total nucleic acid in the sample and comprises genomic DNA and
total RNA including mRNA.
[0062] Preferably, said specific DNA or RNA sequences are chosen
among: [0063] i) human sequences selected from SSAT mRNA (SEQ ID No
62), H3F3A mRNA (SEQ ID No 63) and IL8 mRNA (SEQ ID No 64); and/or
[0064] ii) sequences of bacteria selected from Capnocytophaga
gingivalis (ATCC 33624), Prevotella melaminogenica (ATCC 25845),
Streptococcus mitis (ATCC 15914) and Micrococcus luteus (ATCC
53598D); and/or [0065] iii) the viral sequences of human
papillomavirus, preferably the human papillomavirus 16 (ATCC 45113)
or the human papillomavirus 18 (ATCC 45152).
[0066] Preferably, specific DNA or RNA sequences (also called
nucleic acid sequences) are detected by incubating said genomic DNA
and total RNA with a thermostable enzyme with RNA-dependent Reverse
Transcriptase activity and with DNA-dependent Polymerase
activity.
[0067] More preferably, the combination of RT and PCR is performed
in a single-tube reaction.
[0068] The term "detecting" as used herein is meant to refer to
diagnosing, inferring, evaluating, monitoring, determining the
amount, concentration, ratio, or other quantitative or qualitative
assessment in samples, optionally compared to a control sample, of
nucleic acid and volatile compounds.
[0069] In the context of the present invention, "detecting specific
nucleic acid sequences" comprises comparing the expression level of
the specific nucleic acid sequences to the mean expression level of
said specific nucleic acid sequences in the normal population. A
specific nucleic acid sequence is detected when the expression
level of said specific nucleic acid sequences in the tested saliva
sample is equivalent or superior to 2 fold the mean expression
level of said specific nucleic acid sequences in the normal
population.
[0070] The method of the invention may comprise comparing the level
of expression of specific nucleic acid sequence in a saliva sample
from a subject, with the normal expression level of said nucleic
acid sequence in a control. A significantly higher level of
expression of said gene in the saliva sample of a subject as
compared to the normal expression level is an indication that the
patient has or is predisposed to oral cancer. An "over-expression"
of a specific nucleic acid refers to an expression level in a
saliva sample that is greater than the standard error of the assay
employed to assess expression, and is preferably at least 20%
superior to the normal level of expression of said nucleic acid,
preferably at least 50% superior to the normal level of expression
of said nucleic acid, and most preferably at least 100% superior to
the normal level of expression of said nucleic acid. The "normal"
level of expression of a nucleic acid is the level of expression of
said nucleic acid in a saliva sample of a subject not afflicted
with cancer. Preferably, said normal level of expression is
assessed in a control sample (e.g., sample from a healthy subject,
which is not afflicted by cancer) and preferably, the mean
expression level of said nucleic acid in several control
samples.
[0071] Also, the detection of specific nucleic acid sequences is
based on the detection of at least 50, preferably 70, and even more
preferably 100 successive nucleotides of the specific targeted
nucleic acid sequence as registered in the official data bases, for
example in the NCBI data base.
[0072] For nucleic acid sequences according to ii) as defined
above, "detecting" comprises comparing the expression level to the
expression level in the normal population or to mean expression
level and wherein when expression level is equivalent or superior
of the threshold/cutoff of 10.sup.5 CFU per ml of saliva, it is
indicative of increased oral cancer.
[0073] In one embodiment of the present invention, the detection of
at least two human mRNA sequences as specifically defined in i) and
at least one bacterial sequence as specifically defined in ii)
indicates that the human subject has a high risk of developing an
oral cancer.
[0074] In a preferred embodiment of the present invention, the
detection of at least the human mRNA of H3F3A (SEQ ID No 63) and
the human mRNA of SSAT (SEQ ID No 62) in the saliva sample of a
human subject indicates that said human subject has a high risk
developing an oral cancer.
[0075] In a more preferred embodiment of the present invention, the
detection of the human mRNA of H3F3A (SEQ ID No 63), the human mRNA
of SSAT (SEQ ID No 62) and the bacterial genome of Streptococcus
mitis (ATCC 15914) in the saliva sample of a human subject
indicates that said human subject has a risk superior to 64% of
developing an oral cancer, and has therefore a high risk of
developing an oral cancer.
[0076] On the contrary, when the particulate DNA or RNA sequence as
disclosed in the present invention are not detected in the saliva
sample of a human subject, it means that said human subject has a
low risk of developing an oral cancer.
[0077] As mentioned previously, one problem was to design a test
detecting the various risk factors actually associated with oral
cancer in one simple test reaction. Indeed, some important
biological markers are of different origins, virus, bacteria,
human, or from mRNA or DNA, which makes the detection difficult in
one step reaction test.
[0078] In one embodiment of the present invention, the detection of
nucleic acid sequences comprises incubating said genomic DNA and
total RNA with a thermostable enzyme with RNA-dependent Reverse
Transcriptase activity and with DNA-dependent Polymerase activity,
allowing the combination of RT-PCR and PCR.
[0079] Preferably, the RT-PCR reaction is performed with the Tth
DNA polymerase.
[0080] More preferably, the detection of nucleic acid sequences
comprises incubating said genomic DNA and total RNA in the same
tube with a thermostable enzyme with RNA-dependent Reverse
Transcriptase activity and with DNA-dependent Polymerase activity,
allowing the combination of RT-PCR and PCR in a single-tube
reaction.
[0081] Even more preferably, the detection of nucleic acid
sequences comprises amplifying and detecting at least one DNA or
RNA sequence chosen among SEQ ID No 62 to 70.
[0082] Therefore, in a preferred embodiment, the present invention
is drawn to a method for diagnosing a predisposition to oral
cancer, or for diagnosing an oral cancer in a human subject, the
method comprising the steps of [0083] a) extracting total nucleic
acid (genomic DNA and total RNA) of bacteria, virus, and human
origins from the stabilized saliva sample, [0084] b) incubating
said genomic DNA and total RNA in the same tube with a thermostable
enzyme with RNA-dependent Reverse Transcriptase activity and with
DNA-dependent Polymerase activity, allowing the combination of RT
and PCR in a single-tube reaction, wherein said RT-PCR reaction is
performed with primers and probes specific to: [0085] i) human
sequences selected from SSAT mRNA (SEQ ID No 62), H3F3A mRNA (SEQ
ID No 63) and IL8 mRNA (SEQ ID No 64); [0086] ii) sequences of
bacteria selected from Capnocytophaga gingivalis (ATCC 33624, SEQ
ID No 65), Prevotella melaminogenica (ATCC 25845, SEQ ID No 66),
Streptococcus mitis (ATCC 15914, SEQ ID No 67) and Micrococcus
luteus (ATCC 53598D, SEQ ID No 68), [0087] iii) sequences of virus
selected from human papillomavirus 16 (ATCC 45113, SEQ ID No 69)
and human papillomavirus 18 (ATCC 45152, SEQ ID No 70); and wherein
the presence of at least one sequence of each i), ii) or iii), is
indicative of a risk or a predisposition to oral cancer.
[0088] In a particular embodiment, the presence of at least one
sequence of each i), ii) and iii), is indicative of a risk or a
predisposition to oral cancer.
[0089] More specifically, the detection of the presence of a
combination of at least one of said sequences iii), at least two of
said sequences i) and at least two of said sequences ii) indicates
that the human subject has an increased risk of developing oral
cancer.
[0090] In one particular embodiment, the method comprises
amplifying and detecting SEQ ID No 62 to 70.
[0091] In another particular embodiment, the invention is directed
to a method for risk evaluation and diagnosis of oral cancer
disease in a human subject comprising the steps of:
a) collecting a sample of crude saliva of said human subject in a
sterile device, b) stabilizing said sample by adding a solution
comprising a guanidium salt, such as guanidinum thiocyanate, and/or
ammonium sulfate, and/or sodium azide, and optionally exo and/or
endonuclease inhibitors, c) extracting total nucleic acid of
bacteria, virus, and human origins from the previously obtained
stabilized saliva sample, d) precipitation and purification of
total nucleic acids, e) incubating the purified total nucleic acid
with a thermostable enzyme with RNA-dependant reverse transcriptase
activity and with DNA-dependant polymerase activity and
polynucleotide primers under conditions which allow the reverse
transcriptase activity of said thermostable enzyme to synthetise
cDNA from the ribonucleic and amplification of genomic DNA and cDNA
at a detectable level by Polymerase Chain Reaction, f) detecting in
an assay the amplified DNAs sequences by hybridization with one or
more polynucleotide probes specific to: [0092] i) human sequences
selected from SSAT mRNA (SEQ ID No 62), H3F3A mRNA (SEQ ID No 63)
and IL8 mRNA (SEQ ID No 64); or [0093] ii) sequences of bacteria
selected from Capnocytophaga gingivalis (ATCC 33624, SEQ ID No 65),
Prevotella melaminogenica (ATCC 25845, SEQ ID No 66), Streptococcus
mitis (ATCC 15914, SEQ ID No 67) and Micrococcus luteus (ATCC
53598D, SEQ ID No 68), or [0094] iii) sequences of virus selected
from human papillomavirus 16 (ATCC 45113, SEQ ID No 69) and human
papillomavirus 18 (ATCC 45152, SEQ ID No 70).
[0095] Preferably, said method is performed by detecting the
amplified DNAs sequences by hybridization with one or more
polynucleotide probes specific to i), ii) and iii).
[0096] In the above method, it is also contemplated to add a
calibrated and know nucleic acid (DNA or RNA) to the vial used to
collect saliva to measure the exact quantity of saliva collected by
comparison with an external standard nucleic acid calibration
curve.
[0097] Regarding step d), total nucleic acid present in the
preservative reagent can be purified by a nucleic acid affinity
resin.
[0098] In step e), the purified genomic DNA and total RNA are
incubated in the same tube with a thermostable enzyme with
RNA-dependent Reverse Transcriptase activity and with DNA-dependent
Polymerase activity, allowing the combination of RT and PCR in a
single-tube reaction, such as Tth DNA polymerase, and
polynucleotide primers with nucleotides sequences that detect at
least one of each i), ii) or iii) sequences.
[0099] According to the invention, polynucleotide primers and
probes are natural nucleic acid or Peptide Nucleic Acid (PNA) or
modified nucleic acid (superbase) which can hybridize to nucleic
acid (DNA and RNA).
[0100] Step f) may further comprise the step of quantifying
amplified DNA by comparison with quantified standard DNA or RNA and
determining whether or not the nucleic acid is present, over
present or overexpressed in the saliva sample.
[0101] In one particular embodiment, the invention relates to a
method of measuring the presence of oral cancer risk factors in a
human subject comprising the steps of: [0102] The recovery and
preservation of total nucleic acid from crude saliva from
degradation caused by nucleases, [0103] Optionally, the addition of
a positive nucleic acid control for the full analytic process and
exact quantification of the saliva amount, [0104] Nucleic acid
extraction and concentration of total nucleic acid (genomic DNA and
total RNA), [0105] A one-step Reverse Transcriptase Polymerase
Chain Reaction: The purified genomic DNA and total RNA are
incubated in the same tube with a thermostable enzyme with
RNA-dependent Reverse Transcriptase activity and with DNA-dependent
Polymerase activity, allowing the combination of RT and PCR in a
single-tube reaction, such as Tth DNA polymerase, and
polynucleotide primers with nucleotides sequences that detect
Capnocytophaga gingivalis (ATCC 33624, Genbank Accession AF543295),
Prevotella melaminogenica (ATCC 25845, Genbank Accession No
AJ555137), Streptococcus mitis (ATCC 15914, Genbank Accession No
AJ617805,) Micrococcus luteus (ATCC 53598D, Genbank Accession No
AM285006), human papillomavirus 16 (ATCC 45113, Genbank Accession
No EF422141), human papillomavirus 18 (ATCC 45152, Genbank
Accession No EF422111), SSAT mRNA (Genbank Accession No NM002970),
H3F3A mRNA (Genbank Accession No NM002107), IL8 mRNA (Genbank
Accession No NM000584).
[0106] Using one-step Real-time Reverse Transcriptase Polymerase
Chain Reaction, the invention enables the user to perform a rapid
RT-PCR and simultaneously detect and quantify the presence of total
nucleic acid from bacteria, virus and human mRNA by monitoring
fluorescence during real time polymerase chain reaction
amplification without any risk of false positive due to opening
tube between RT and PCR and from possible PCR product environmental
contamination due to precedent amplification reactions in the same
environment.
[0107] Beta actine mRNA (Genbank Accession Number X00351--SEQ ID No
61) is useful as internal standard in step a) for calibration and
quantification.
[0108] In a preferred embodiment, the polymerase with RNA-dependent
Reverse Transcriptase activity and with DNA-dependent Polymerase
activity is the Tth DNA polymerase.
[0109] Using total nucleic acid extraction and one-step Real-time
Reverse Transcriptase Polymerase Chain Reaction in a same
microfluidic cartridge, the invention enables the user to perform a
point of care analysis usable in physician and dentist offices.
[0110] Alternatively, using one-step Reverse Transcriptase
Polymerase Chain Reaction and a microarray, the invention enables
the user to perform RT-PCR and detect and quantify the presence of
total nucleic acid from bacteria, virus and human mRNA by measuring
hybridization signal at the end of the RT-PCR and compare with
external nucleic standard.
[0111] In a specific embodiment, the present invention is drawn to
a micro array for diagnosing a predisposition to oral cancer in a
human subject comprising probes sequences selected from the group
consisting of SEQ ID No 32 to SEQ ID No 60.
[0112] In this aspect of the present invention, the polynucleotide
primers and probes to amplify and detect at least one of each i),
ii) or iii) sequences are preferably selected from the group
consisting of SEQ ID No:1 to SEQ ID No:60.
[0113] Kit of parts associated with the methods herein disclosed
are also disclosed. In an exemplary embodiment, the present
invention therefore relates to a kit to practice the above method,
comprising primers and probes sequences to amplify and detect at
least one of each of the sequences i), ii) or iii) depicted above,
and including at least one of the following:
a) a sterile device to collect a saliva sample, optionally
including a control nucleic acid, and a collect reagent, b) a
preservation reagent, for example a spray dry preservative reagent,
c) a resin having affinity for total nucleic acid, d) a
thermostable enzyme with RNA-dependant reverse transcriptase
activity and with DNA-dependant polymerase activity and
polynucleotide.
[0114] As mentioned before, the collect reagent is a dilution
buffer which is preferably citrate buffer.
[0115] The kit comprises a preservation reagent, which is
preferably a buffer comprising a salt such as guanidinum
thiocyanate, and/or ammonium sulfate and/or sodium azide, and
optionally exo and/or endonuclease inhibitors. The sodium azide
salt is employed preferably at a concentration range between 20 mM
and 100 mM, more preferably around 40 mM. Other salts, such as
guanidium thiocyanate and/or ammonium sulphate, are added at a
concentration of 4M.
[0116] In one embodiment of the present invention, the preservation
reagent is provided in dry format in a sterile plastic tube under
vacuum, which can draw up the crude saliva associated with the
dilution buffer.
[0117] Primers and probes sequences to amplify and detect at least
one of each of the sequences i), ii) or iii) depicted above are
further defined as comprising polynucleotide primers for
synthesizing cDNA by reverse transcription, polynucleotide primers
for amplifying genomic DNA and cDNA by polymerase chain reaction
and polynucleotide probes for detecting amplified DNA.
[0118] In the diagnostic kits herein disclosed, the reagent can be
provided in the kits, with suitable instructions and other
necessary reagents in order to perform the methods here disclosed.
Instructions, for example written or audio instructions, on paper
or electronic support such as tapes or CD-roms, for carrying out
the assay, will be usually included in the kit.
[0119] In a second aspect, the present invention is drawn to a
method of diagnosing a predisposition to oral cancer in a human
subject, the method comprising collecting and stabilizing a crude
saliva sample from said human subject, and analyzing a volatile
fraction extracted from said stabilized saliva by detecting in said
volatile fraction at least one biochemical organic compound,
wherein the detection of at least one biochemical organic compound
is indicative of a risk or a predisposition to oral cancer.
[0120] The present invention is also drawn to an in vitro method of
diagnosing oral cancer in a human subject, the method comprising
stabilizing a crude saliva sample from said human subject, and
analyzing a volatile fraction extracted from said stabilized saliva
by detecting in said volatile fraction at least one biochemical
organic compound, wherein the detection of at least one biochemical
organic compound is indicative of a risk of developing an oral
cancer.
[0121] From a chemistry point of view, biochemical organic
compounds are the members of a large class of chemical compounds
whose molecules contain carbon. They can be antigens,
carbohydrates, enzymes, hormones, lipids, fatty acids,
neurotransmitters, nucleic acids, proteins, peptides and amino
acids, vitamins, fats and oils.
[0122] Among all the known organic compounds, "Volatile organic
compounds" (VOC) are meant to designate any organic compound that
is volatile, i.e. that have a high vapor pressure or low boiling
point, and can therefore evaporate at normal temperature and
pressure. These compounds are often regulated by governments. For
example, in European Union, a "Volatile Organic Compound" is any
organic compound having an initial boiling point less than or equal
to 250.degree. C. measured at a standard atmospheric pressure of
101.3 kPa.
[0123] In the context of the invention, the "volatile fraction" is
recovered from the heating of a crude saliva sample. Preferably,
said volatile fraction is extracted from crude saliva sample by
heating said saliva sample for at least 10 minutes, preferably 20
minutes and more preferably 30 minutes at a temperature comprised
between 30.degree. C. and 50.degree. C., preferably 40.degree. C.
During this time, the volatile fraction is taken away from the
sample by using a solid-phase microextraction (SPME) with a
carboxen/polydimethylsiloxane coated fiber (CAR/PDMS fiber).
[0124] Therefore, in one embodiment of the present invention, the
volatile biochemical organic compounds are extracted with a
CAR/PDMS fiber coating during at least, preferably 20 minutes, and
even more preferably 30 minutes from a saliva sample that is
simultaneously heated at a temperature comprised between 30.degree.
C. and 50.degree. C., and preferably at about 40.degree. C. The
desorption temperature of the fiber is comprised between
250.degree. C. and 300.degree. C., and is preferably of about
280.degree. C.
[0125] Solid-phase microextraction (SPME) is a patented sample
preparation technique based on the adsorption of analytes directly
from an aqueous sample onto a coated, fused-silica fiber. This
sampling technique is fast, easy to use and eliminates the use of
organic solvents (Mills G et al, Journal of Chromatography 2000;
Song C et al, Lung Cancer 2009).
[0126] In this technology, the CAR/PDMS fibers are often used for
detecting trace level of volatile compounds, and are therefore
well-known from the man skilled in the art (Garcia-Esteban M et al,
Talanta 2004).
[0127] Preferably, the detection of said biochemical organic
compound is performed by using a chromatograph in gas phase coupled
to a mass spectrometer.
[0128] In the context of the invention, a biochemical compound is
"detected" when the expression level of said compound is at least
superior to 1.5 fold the mean expression level of said compound in
the normal population.
[0129] By applying the method of the invention, some biochemical
compounds were shown to be highly overexpressed in the volatile
fraction of human subjects suffering from oral cancer and were
therefore found to be acute and sensitive diagnostic and/or
prognostic tool of oral cancer. Importantly, none of these
compounds can be detected in the fluid fraction of saliva,
highlighting the necessity to study the volatile fraction of saliva
in this case.
[0130] In a preferred embodiment, the present invention is drawn to
a method of diagnosing a risk or a predisposition to oral cancer in
a human subject by analyzing the content in biochemical compounds
in the volatile fraction of stabilized samples of saliva, wherein
the detection of at least one of the biochemical organic compound
selected in the group consisting of: 2,3-pentanedione (CAS number
600-14-6), 3-methyltiophene (CAS number 616-44-4), acetone (CAS
number 67-64-1), hexanenitrile (CAS number 628-73-9), benzaldehyde
(CAS number 100-52-7), 3-methyl-2-pentanone (CAS number 565-61-7),
2,3-butanedione (CAS number 431-03-8), 2-propanol (CAS number
67-63-0), ethyl acetate (CAS number 141-78-6), 1-propanol (CAS
number 71-23-8), hexanal (CAS number 66-25-1),
5-methyl-3-hexen-2-one (CAS number 5166-53-0), m-xylene (CAS number
108-38-3), p-xylene (CAS number 106-42-3), 2-methyl-2-butenal (E)
(CAS number 497-03-0), phenol (CAS number 108-95-2), butanal (CAS
number 123-72-8), methylbutanone (CAS number: 563-80-4),
2-methyl-2-butene (CAS number 513-35-9), 2-methyl-1-propene (CAS
number 115-11-7) and (cis) 1,2 dimethyl-cyclopropane (CAS number:
930-18-7) is indicative of a risk or predisposition to oral
cancer.
[0131] Indeed, the below-presented results have shown that the
detection of at least one of the following compounds:
2,3-pentanedione (CAS number 600-14-6), 3-methyltiophene (CAS
number 616-44-4), acetone (CAS number 67-64-1), hexanenitrile (CAS
number 628-73-9), benzaldehyde (CAS number 100-52-7),
3-methyl-2-pentanone (CAS number 565-61-7), 2,3-butanedione (CAS
number 431-03-8), 2-propanol (CAS number 67-63-0), ethyl acetate
(CAS number 141-78-6), 1-propanol (CAS number 71-23-8), hexanal
(CAS number 66-25-1), 5-methyl-3-hexen-2-one (CAS number
5166-53-0), m-xylene (CAS number 108-38-3), p-xylene (CAS number
106-42-3), 2-methyl-2-butenal (E) (CAS number 497-03-0) in the
volatile fraction of saliva of a human subject indicates that said
human subject has a high risk of being predisposed to develop oral
cancer or has a high risk of being developing an oral cancer. More
precisely, the detection of at least one of the above-mentioned
biochemical organic compounds indicates that the tested human
subject has a risk superior to 60% of developing an oral cancer;
the detection of at least two of the above-mentioned biochemical
organic compounds indicates that the tested human subject has a
risk superior to 70% of developing an oral cancer, and the
detection of at least three of the above-mentioned biochemical
organic compounds indicates that the tested human subject has a
risk superior to 80% of developing an oral cancer; the detection of
at least four of the above-mentioned biochemical organic compounds
indicates that the tested human subject has a risk superior to 90%
of developing an oral cancer. On the contrary, when at least one of
the particulate biochemical organic compounds disclosed above is
not detected in the saliva sample of a human subject, it means that
said human subject has a low risk of developing an oral cancer.
[0132] For example, as shown in the example 10, the detection of
the biochemical organic compounds of the group comprising:
hexanitrile, 2,3-pentanedione, 3-methylthiophene and acetone in the
volatile fraction of saliva of a human subject indicates that said
human subject has a risk superior to 97% of developing an oral
cancer, and is therefore predisposed to develop an oral cancer, or
is developing an oral cancer.
[0133] Also, as shown in the example 11, the detection of the mRNA
sequence of H3F3A (SEQ ID No 63), SSAT (SEQ ID No 62) and of the
biochemical organic compounds hexanenitrile, 2,3-pentanedione,
3-methylthiophene and acetone in the saliva of a human subject
enables to detect 100% of cancer cases in a tested population.
Therefore, in a preferred embodiment, the detection of the mRNA
sequence of H3F3A (SEQ ID No 63), SSAT (SEQ ID No 62) and of the
biochemical organic compounds hexanenitrile, 2,3-pentanedione,
3-methylthiophene and acetone in the saliva of a human subject
indicates that said human subject is developing an oral cancer.
[0134] Also, as examplified hereunder, the method of the present
invention can rely on the detection of 3-methyl-2-pentanone, methyl
butanone, butanal, hexanal, hexanenitrile, 1-propanol 2-propanol,
(cis) 1,2-dimethyl cyclopropane, phenol, and 2,3-butanedione in
order to prognose or to diagnose an oral cancer in a human
subject.
[0135] In another embodiment, the present invention is drawn to a
method of diagnosing a risk or a predisposition to oral cancer in a
human subject, wherein the detection of at least one biochemical
organic compound in the volatile fraction of saliva of a human
subject indicates that said human subject is not predisposed to
develop an oral cancer or is not developing an oral cancer.
[0136] The compounds 2-methyl-2-butene (CAS number 513-35-9),
2-methyl-1-propene (CAS number 115-11-7) and (cis) 1,2-dimethyl
cyclopropane (CAS number 930-18-7) are overexpressed in the
volatile fraction of healthy human subject and are absent in the
volatile fraction of patients suffering from oral cancer.
Therefore, these compounds can serve as "healthy biochemical
markers". The detection of at least one, preferably two of these
biochemical organic compounds indeed indicates that the human
subject has a low risk of being predisposed of being developing an
oral cancer.
[0137] Moreover, the detection of particulate biochemical organic
compounds such as benzaldehyde, acetone, 2,3-pentanedione, on a one
hand, and the absence of other particulate biochemical compounds
such as 2-methyl-2-butene, 2-methyl-1-propene, and/or (cis)
1,2-dimethyl cyclopropane on the other hand, enables to diagnose
oral cancer with a high sensitivity (at least 93%, see example
10).
[0138] It is noteworthy that most of these biochemical compounds
have never been identified so far in the saliva. Moreover, none of
them have been related so far with cancer predisposition, and a
fortiori with oral cancer predisosition.
[0139] Therefore, the present invention is also drawn to the use of
the detection of at least one compound chosen among:
2,3-pentanedione (CAS number 600-14-6), 3-methyltiophene (CAS
number 616-44-4), acetone (CAS number 67-64-1), hexanenitrile (CAS
number 628-73-9), benzaldehyde (CAS number 100-52-7),
3-methyl-2-pentanone (CAS number 565-61-7), 2,3-butanedione (CAS
number 431-03-8), 2-propanol (CAS number 67-63-0), ethyl acetate
(CAS number 141-78-6), 1-propanol (CAS number 71-23-8), hexanal
(CAS number 66-25-1), 5-methyl-3-hexen-2-one (CAS number
5166-53-0), m-xylene (CAS number 108-38-3), p-xylene (CAS number
106-42-3), 2-methyl-2-butenal (E) (CAS number 497-03-0),
2-methyl-2-butene (CAS number 513-35-9), 2-methyl-1-propene (CAS
number 115-11-7) and (cis) 1,2-dimethyl cyclopropane (CAS number
930-18-7) in a diagnostic test to assess the risk of developing an
oral cancer in a human subject.
[0140] In a particulate embodiment, the present invention is
therefore drawn to a method of diagnosing a risk or a
predisposition to oral cancer in a human subject, comprising the
steps of: [0141] a) collecting a sample of crude saliva of said
human subject in a sterile device, [0142] b) stabilizing said
sample by adding a solution comprising a salt, such as guanidinum
thiocyanate, ammonium sulfate and/or sodium azide, [0143] c)
extracting the volatile fraction from said stabilized sample by
heating it for at least 10 minutes at 40.degree. C. and using for
example Solid-phase Microextraction (SPME) to take away the
volatile fraction, [0144] d) detecting at least one biochemical
organic compound by using for example a chromatograph in gas phase
coupled to a mass spectrometer, wherein the detection of at least
one, preferably at least two, and more preferably at least three
biochemical organic compound(s) is indicative of a risk or a
predisposition to oral cancer.
[0145] In a preferred embodiment, the at least one, preferably at
least two, and more preferably at least three detected biochemical
organic compound is (are) chosen among: 2,3-pentanedione (CAS
number 600-14-6), 3-methyltiophene (CAS number 616-44-4), acetone
(CAS number 67-64-1), hexanenitrile (CAS number 628-73-9),
benzaldehyde (CAS number 100-52-7), 3-methyl-2-pentanone (CAS
number 565-61-7), 2,3-butanedione (CAS number 431-03-8), 2-propanol
(CAS number 67-63-0), ethyl acetate (CAS number 141-78-6),
1-propanol (CAS number 71-23-8), hexanal (CAS number 66-25-1),
5-methyl-3-hexen-2-one (CAS number 5166-53-0), m-xylene (CAS number
108-38-3), p-xylene (CAS number 106-42-3) and, 2-methyl-2-butenal
(E) (CAS number 497-03-0), phenol (CAS number 108-95-2), butanal
(CAS number 123-72-8), methylbutanone (CAS number: 563-80-4),
2-methyl-2-butene (CAS number 513-35-9), 2-methyl-1-propene (CAS
number 115-11-7) and (cis) 1,2 dimethyl-cyclopropane (CAS number:
930-18-7).
[0146] In a preferred embodiment, the detection of the at least one
detected biochemical compound chosen among 2,3-pentanedione (CAS
number 600-14-6), 3-methyltiophene (CAS number 616-44-4), acetone
(CAS number 67-64-1), hexanenitrile (CAS number 628-73-9),
benzaldehyde (CAS number 100-52-7), 3-methyl-2-pentanone (CAS
number 565-61-7), 2,3-butanedione (CAS number 431-03-8), 2-propanol
(CAS number 67-63-0), ethyl acetate (CAS number 141-78-6),
1-propanol (CAS number 71-23-8), hexanal (CAS number 66-25-1),
5-methyl-3-hexen-2-one (CAS number 5166-53-0), m-xylene (CAS number
108-38-3), p-xylene (CAS number 106-42-3), and 2-methyl-2-butenal
(E) (CAS number 497-03-0) is indicative of a high risk of
developing cancer as compared to a normal healthy population.
[0147] On the contrary, when at least one of the biochemical
compounds: 2-methyl-2-butene (CAS number 513-35-9),
2-methyl-1-propene (CAS number 115-11-7) or (cis) 1,2-dimethyl
cyclopropane (CAS number 930-18-7) is detected, it is indicative of
a poor risk of developing oral cancer, i.e. a risk inferior to 10%,
preferably 5% to develop cancer as compared to a normal healthy
population.
[0148] In another embodiment, the present invention is drawn to a
kit to practice a method of diagnosing a risk or a predisposition
to oral cancer based on the volatile fraction of saliva,
comprising: [0149] a) A sterile device to collect a saliva sample,
optionally containing a collect reagent [0150] b) A preservation
reagent, [0151] c) At least one electronic sensor, [0152] d)
Optionally, a control molecular marker.
[0153] As mentioned before, the collect reagent is a dilution
buffer which is preferably a citrate buffer.
[0154] The kit comprises a preservation reagent, which is
preferably a buffer comprising a salt capable of reducing the
vapour tension of volatile compounds without allowing the
degradation of said compounds. This salt is preferably a salt such
as guanidinum thiocyanate, and/or ammonium sulfate and/or sodium
azide. This sodium azide is employed preferably at a concentration
range between 20 mM and 6M, preferably between around 10 mM and 100
mM, more preferably around 40 mM. Other salts, such as guanidium
thiocyanate and/or ammonium sulphate, are added at a concentration
of 4M.
[0155] In one embodiment of the present invention, the preservation
reagent is provided in a dry format in a sterile plastic tube under
vacuum, which can draw up the saliva associated with the dilution
buffer.
[0156] In the context of the invention, the device used to detect
the organic compounds in the collected volatile fraction of saliva
is an electronic sensor, for example electronic noses, JPL
electronic noses, FET-type Bioelectronic noses, alpha mos). These
technologies are now widely used and therefore known from the man
skilled in the art (Cho S. M., Sensors and Actuators 2006).
[0157] Using specific electronic sensors for the identification of
the targeted volatile compounds in a specific platform (electronic
nose), the invention enables the user to perform a specific
analysis platform or a point of care analysis usable in physician
and dentists offices.
[0158] In one embodiment of the invention the control molecular
markers are chosen among: 1-bromobutane, 1-bromobenzene and
1,4-dibromobenzene.
Example 1
Stabilization of Crude Samples of Saliva
[0159] Raw saliva is collected with a medical device which makes
easier the collection of a large volume of saliva (up to 2 ml)
following by a stabilization of the saliva biomarkers (AND, ARN,
peptides, volatile compound).
[0160] 4 ml of saliva extraction solution is then swallowed up to 2
minutes for collection of 2 ml of saliva.
[0161] The saliva extraction solution contains: [0162] FD&C
yellow no 5 (tartrazine) [0163] Citrate buffer (39 mM)
[0164] The 2 ml of diluted saliva (in 4 ml) are then transferred in
tubes containing lyophilized sodium azide for biomarker
stabilization. The final sodium azide concentration is about 40
mM.
[0165] 2 tubes are prepared for each sample. A tube is intended for
the "genetic" analyzes (tube 1) and the other tube (tube 2) is used
for the analyzis of the volatile compounds.
[0166] The tubes can be transported without control of temperature
during 10 days before being analyzed.
Example 2
Simultaneous Determination and Quantification in Stabilized Saliva
of the Presence of Bacteria, Virus and Human mRNA to Assess Risk
Factor of Oral Cancer
[0167] 0.250 to 3 ml of the solution from tube 1 is prepared and
total nucleic acids are extracted by centrifiltration on a silica
membrane without DNAse step. Up to 1.5 mg of total nucleic acid are
extracted and purified from 1 ml of stabilized saliva.
[0168] Isolation of high-quality DNA and RNA from whole saliva
samples is difficult because under ambient conditions, expression
and quantification profile are unstable on a timescale below few
minutes. This instability is the result of metabolic activity of
bacteria, nutrients dependant, concentration in nucleases and
limited turnover of RNA in that environmental conditions. In order
to render the nucleic acid inaccessible to nuclease, we used a
preservation buffer which comprises a salt for membrane lysis of
bacteria as well as human cells and precipitates the extracted
nucleic acid in the sample along with the cellular protein. We used
in this regard a guanidinium salt such as guanidinum thiocyanate
and or ammonium sulfate associated or not with a ribonuclease
inhibitor (EDTA<10 mM).
[0169] Preferred biological targets to be detected in crude saliva
are Capnocytophaga gingivalis (ATCC 33624, Genbank Accession No
AF543295), Prevotella melaminogenica (ATCC 25845, Genbank Accession
No AJ555137), Streptococcus mitis (ATCC 15914, Genbank Accession No
AJ617805,) Micrococcus luteus (ATCC 53598D, Genbank Accession No
AM285006), human papillomavirus 16 (ATCC 45113, Genbank Accession
No EF422141), human papillomavirus 18 (ATCC 45152, Genbank
Accession No EF422111), SSAT mRNA (Genbank Accession No NM002970),
H3F3A mRNA (Genbank Accession No NM002107), IL8 mRNA (Genbank
Accession No NM000584).
[0170] Beta actine mRNA (Genbank Accession No X00351) is used as
internal control for calibration and quantification.
[0171] After obtaining bacteria, virus, human cells and
extracellular human total nucleic acid from a saliva sample in a
sterile device, the preservation buffer is added into the saliva
sample at room temperature. The buffer is preferably able to open
prokaryotic and eukaryotic cells, associated with preservation of
total nucleic acids by action of blocking nucleases activities and
precipitation of total nucleic acids. The preservation reagent is
provided in dry format in a sterile polyethylene terephtalate (PET)
plastic tube. Stabilizing reagent is calibrated to draw up to 3 ml
of saliva associated with a dilution buffer. Calibrated and known
nucleic acids (DNA or RNA), for example can be added to the collect
vial in order to measure the exact quantity of saliva collected and
analyzed by comparison with external standard calibration curve
obtained after extraction and Reverse Transcriptase PCR
quantification (some of the biotargets measured are expressed in
genomic quantity per ml of saliva). Calibrated and known nucleic
acids (DNA or RNA) added to the collect vial will also permit to
verify the global performance of the full analytical process. Total
nucleic acids are purified with a Nucleic Acid affinity resin. Our
preferred system used coated paramagnetic beads compatible with the
guanidinium salt having a rate of recovery of total nucleic acids
from crude sample up to 90% and no selection between RNA and
DNA.
[0172] The basic RT PCR process is carried out as follows. The RNA
present in the total nucleic acid contained in the sample may be
first reverse transcribed into cDNA (using enzyme like Tth DNA
polymerase as purified enzyme and a oligonucleotide or PNA or
modified oligonucleotide), and then denatured, using physical
means, which are known to those of skill in the art. A preferred
physical means for strand separation involves heating the nucleic
acid until it is completely (>99%) denatured. Methods for the
amplification of RNA targets using a thermostable DNA polymerase
are described in WO9109944 incorporated herein by reference. The
denatured DNA strands are then incubated in the same tube with the
selected oligonucleotide primers under hybridization conditions,
conditions which enable the binding of the primers to the single
DNA strands. As known in the art, the primers are selected so that
their relative positions along a duplex sequence are such that an
extension product synthesized from one primer, when it is separated
from its complement, serves as a template for the extension of the
other primer to yield a replicate chain of defined length. The
primer must be sufficiently long to prime the synthesis of
extension products in the presence of the agent for polymerization.
The exact length of the primers will depend on many factors,
including temperature, source of the primer and use of the
method.
[0173] Preferred oligonucleotide primers for use in the present
invention are selected from the group consisting of SEQ ID No 1 to
SEQ ID No 31.
[0174] Template-dependent extension of the oligonucleotide primer
(s) is then catalyzed by the polymerizing agent (in the presence of
adequate amounts of the four deoxyribonucleoside triphosphates
(dATP, dGTP, dCTP, and dTTP or analogs), in a reaction medium which
is comprised of the appropriate salts, metal cations, and pH
buffering system. The products of the synthesis are duplex
molecules consisting of the template strands and the primer
extension strands, which include the target sequence. These
products, in turn, serve as templates for another round of
replication. In the second round of replication, the primer
extension strand of the first cycle is annealed with its
complementary primer; synthesis yields a "short" product which is
bounded on both the 5'- and the 3'-ends by primer sequences or
their complements. Repeated cycles of denaturation, primer
annealing, and extension result in the exponential accumulation of
the target region defined by the primers. Sufficient cycles are run
to achieve the desired amount of polynucleotide containing the
target region of nucleic acid. The desired amount may vary, and is
determined by the function which the product polynucleotide is to
serve.
[0175] The PCR method is performed in a fashion where all of the
reagents are added simultaneously, in one step. In a preferred
method, the RT PCR reaction is carried out as an automated process
which utilizes a thermostable enzyme like Tth. In a preferred
method, the RT PCR reaction is performed in a types of thermocycler
having capability for reading at least 4 different florescence dyes
and developed/manufactured for real time PCR assays and commercial
use.
[0176] Those skilled in the art will also be aware of the problems
of contamination of a PCR by the nucleic acid from bacteria
previously present in water used for buffer and resulting in non
specific amplification or background. Methods to reduce these
problems are provided by using adequate buffer, reagents and
enzymes to avoid nucleic acid strand fragments with a size higher
than 100 bp. All reagents used in the RT PCR reaction have to be
processed before using. During amplification by PCR, the target
polynucleotides may be detected directly by hybridization with a
probe polynucleotide which forms a stable hybrid with the target
sequence under high stringency to low stringency hybridization and
washing conditions. Probes are typically labeled with
non-radioactive labeling systems, such as fluoresceins and
derivated systems.
[0177] Reverse Transcriptase activity and with DNA-dependent
Polymerase activity, allowing the combination of RT and PCR in a
single-tube reaction, such as Tth DNA polymerase or an enzyme like
Tth DNA polymerase, and polynucleotide primers with a nucleotide
sequence selected from the group consisting of SEQ ID No 2 SEQ ID
No 4 SEQ ID No 5 SEQ ID No 8 SEQ ID No 10 SEQ ID No 12 SEQ ID No 14
SEQ ID No 16 SEQ ID No 18 SEQ ID No 20 SEQ ID No 21 SEQ ID No 23
SEQ ID No 25 SEQ ID No 27 SEQ ID No 29 and SEQ ID No 31 under
conditions which allow hybridization of the polynucleotide to the
ribonucleotide target region and Reverse Transcriptase activity of
the said polymerase, or enzyme like Tth, for cDNA synthesis; and
(c) amplified the cDNAs formed to a detectable level by Polymerase
Chain Reaction with said polymerase enzyme like Tth DNA polymerase
and polynucleotide primers and probes with a nucleotide sequence
selected from the group consisting of SEQ ID No 1 to SEQ ID No
60.
[0178] More particularly, the preferred combination of primers and
probes used to detect bacteria, virus and human mRNA consists of
the sequences:
Prevotella melaminogenica (ATCC 25845, Genbank Accession No AJ
555137)
Seq ID No 1+Seq ID No 2+Seq ID No 32, or Seq ID No 1+Seq ID No
2+Seq ID No 49, or Seq ID No 3+Seq ID No 2+Seq ID No 49, or Seq ID
No 1+Seq ID No 4+Seq ID No 32, or Seq ID No 3+Seq ID No 5+Seq ID No
49
[0179] Streptococcus mitis (ATCC 15914, Genbank Accession No
AJ617805,)
Seq ID No 6+Seq ID No 8+Seq ID No 50 or Seq ID No 7+Seq ID No 8+Seq
ID No 33 or Seq ID No 7+Seq ID No 8+Seq ID No 51
[0180] Capnocytophaga gingivalis (ATCC 33624, Genbank Accession
AF543295)
Seq ID No 9+Seq ID No 10+Seq ID No 34 or Seq ID No 9+Seq ID No
10+Seq ID No 52
[0181] Micrococcus luteus (ATCC 53598D, Genbank Accession No
AM285006)
Seq ID No 11+Seq ID No 12+Seq ID No 35, or Seq ID No 11+Seq ID No
12+Seq ID No 53
Human Papillomavirus 16 (ATCC 45113, Genbank Accession No
EF422141),
Seq ID No 13+Seq ID No 14+Seq ID No 36
Human Papillomavirus 18 (ATCC 45152, Genbank Accession No
EF422111)
Seq ID No 15+Seq ID No 16+Seq ID No 37
[0182] Beta Actine mRNA (Genbank Accession No X00351) Seq ID No
17+Seq ID No 18+Seq ID No 38 or Seq ID No 17+Seq ID No 18+Seq ID No
39 or Seq ID No 17+Seq ID No 18+Seq ID No 54 or Seq ID No 17+Seq ID
No 18+Seq ID No 55 or Seq ID No 19+Seq ID No 20+Seq ID No 40 or Seq
ID No 19+Seq ID No 20+Seq ID No 41 or Seq ID No 19+Seq ID No 20+Seq
ID No 56 SSAT mRNA (Genbank Accession No NM002970)
Seq ID No 22+Seq ID No 23+Seq ID No 42 or Seq ID No 22+Seq ID No
23+Seq ID No 57 or Seq ID No 24+Seq ID No 25+Seq ID No 43
[0183] H3F3A mRNA (Genbank Accession No NM002107
Seq ID No 26+Seq ID No 27+Seq ID No 44 or Seq ID No 26+Seq ID No
27+Seq ID No 58 or Seq ID No 26+Seq ID No 27+Seq ID No 45 or Seq ID
No 28+Seq ID No 29+Seq ID No 46 or Seq ID No 28+Seq ID No 29+Seq ID
No 59
[0184] IL8 mRNA (Genbank Accession No NM000584)
Seq ID No 30+Seq ID No 31+Seq ID No 47 or Seq ID No 30+Seq ID No
31+Seq ID No 48 or Seq ID No 30+Seq ID No 31+Seq ID No 60
[0185] Also, according to the invention, the preferred probes used
to detect bacteria, virus and human mRNA fixed on a microarray
surface are selected from the group consisting of SEQ ID No 32 to
SEQ ID No 60.
Example 3
Analysis of Genetic Markers in the Fluid Fraction by More than 2
Separate Steps
[0186] 1--The saliva sample (up to 1000 .mu.L) is mixed with a
diluting buffer (sterile nuclease free reagent) and passed through
a sterile polyethylene terephtalate (PET) plastic tube.
Preservative reagent and a known nucleic acid (pure synthetic
ribonucleotide) are calibrated to draw up to 3 ml of saliva
associated with the dilution buffer. Full process should be
realized in less than 2 minutes. This process permits immediate
preservation of total nucleic acids at room temperature for up to
10 days to allow transportation delays via regular mail to
laboratory.
[0187] 2--Lysis at laboratory, transfer up to 1000 .mu.l of liquid
from the PET plastic into a 2 mL sterile tube with up to 1 mL of
lysis buffer and then incubate at 35.degree. C.+/-2.degree. C. for
up to one hour.
[0188] 3--The lysat is processed for total nucleic acids
purification with magnetic silica or polystyrene beads or
funnel-design having silica membrane in mini prep spin columns able
to concentrate circulating nucleic acid from plasma. The elution
volume is up to 100 .mu.l. 5-2 .mu.L (up to 5 .mu.L) of pure
nucleic acids extract is used for the one step real time RT-PCR
(RotorGene) with enzyme like Tth and the following program with
Taqman Probe: I: Reverse transcription 61.degree. C./20 min
(20.degree. C./sec) II: Denaturation 95.degree. C./30 secondes
(20.degree. C./sec) III: PCR (35 cycles) 95.degree. C./5 seconds
(20.degree. C./sec) 60.degree. C./30 seconds (20.degree. C./sec).
The emitted fluorescence is measured at the end of the 60.degree.
C.
Example 4
Analysis of the Fluid Fraction of Saliva Sample Using
Microarrays
[0189] 1--The saliva sample (up to 1000 .mu.L) is mixed with a
diluting buffer (sterile nuclease free water) and passed through a
sterile polyethylene terephtalate (PET) plastic tube. Preservative
reagent and a known nucleic acid (pure synthetic ribonucleotide)
are calibrated to draw up to 3 ml of saliva associated with the
dilution buffer. Full process should be realized in less than 2
minutes. This process permits immediate preservation of total
nucleic acid at room temperature for up to 10 days to allow
transportation delays via regular mail to laboratory.
[0190] 2--Lysis at laboratory, transfer up to 1000 .mu.l of liquid
from the PET plastic into a 2 mL sterile tube with up to 1 mL of
lysis buffer and then incubate at 35.degree. C.+/-2.degree. C. for
up to one hour.
[0191] 3--The lysat is processed for total nucleic acids
purification with magnetic silica or polystyrene beads or
funnel-design having silica membrane in mini prep spin columns able
to concentrate circulating nucleic acid from plasma. The elution
volume is up to 100 .mu.l. 5-2 .mu.L (up to 5 .mu.L) of pure
nucleic acids extract is used for the one step RT-PCR with enzyme
like Tth and the following program without Probes: I: Reverse
transcription 61.degree. C./20 min (20.degree. C./sec) II:
Denaturation 95.degree. C./30 secondes III: PCR (35 cycles)
95.degree. C./20 seconds, 60.degree. C./20 seconds, 72.degree.
C./30 seconds.
[0192] Primers are Cy5 fluorescence labeled. Probes have been
coated on surface of a PET slide using oligonucleotide arms.
[0193] 5 .mu.l of amplified DNA is mixed in 30 .mu.L of
hybridization buffer and incubated 2 minutes at 95.degree. C. 25
.mu.l of the denatured amplified DNA is hybridized on the
microarray at room temperature during 10 minutes, briefly washed in
two washing buffer. The microarray is dried and fluorescence is
measured on a scanner and compared to standards.
Example 5
Analysis of the Fluid Fraction of Saliva by Using Real Time PCR
Point of Care Instrument Associated to a Microfluidics Cartridge
Able to Extract and Amplify Total Nucleic Acids (Genexpert.TM.
Solution from Cepheid Inc or Liat.TM. System from Iquum Inc).
[0194] 1--The saliva sample (up to 1000 .mu.L) is mixed with a
diluting buffer (sterile nuclease free water) and passed through a
sterile polyethylene terephtalate (PET) plastic tube. Stabilizing
reagent and a know nucleic acid (pure synthetic ribonucleotide) are
calibrated to draw up to 3 ml of saliva associated with the
dilution buffer.
[0195] Full process should be realized in less than 2 minutes.
[0196] This process permits immediate preservation of total nucleic
acid at room temperature for up to 10 days to allow transportation
delays via regular mail to laboratory or direct analysis in the
dental of physician office.
[0197] 2--Up to 3 ml or 5 ml of saliva in stabilizing reagent is
transfered in the microfluidics cartridge chamber having lysis
buffer able to process for total nucleic acid purification with
polystyrene beads and/or associated to physical and/or mechanical
lysis. Without technician intervention, 2 .mu.L (up to 5 .mu.L) of
pure nucleic acids extracted are transferred in the one step real
time RT-PCR chamber having enzyme like Tth and associated mix PCR
reagents and the following program [0198] with Taqman Probe: I:
Reverse transcription 61.degree. C./20 min (20.degree. C./sec) II:
Denaturation 95.degree. C./30 seconds (20.degree. C./sec) III: PCR
(35 cycles) 95.degree. C./5 seconds (20.degree. C./sec) 60.degree.
C./30 seconds (20.degree. C./sec). The emitted fluorescence is
measured at the end of the 60.degree. C.
Example 6
Evaluation of the Overexpression of Several Genetic Marker in the
Saliva of Oral Cancer Patients by Quantitative PCR
[0199] 1--The saliva sample (up to 1000 .mu.L) is mixed with a
diluting buffer (sterile nuclease free water) and passed through a
sterile polyethylene terephtalate (PET) plastic tube. Preservative
reagent and a known nucleic acid (pure synthetic ribonucleotide)
are calibrated to draw up to 3 ml of saliva associated with the
dilution buffer. Full process should be realized in less than 2
minutes. This process permits immediate preservation of total
nucleic acid at room temperature for up to 10 days to allow
transportation delays via regular mail to laboratory.
[0200] 2--Lysis at laboratory, transfer 250 .mu.l of liquid from
the PET plastic into a 2 mL sterile tube with Proteinase K and then
incubate at 56.degree. C.+/-2.degree. C. for up to one hour.
Incubate the pre-lysed sample with a lysis buffer, 10 minutes at
70.degree. C.+/-2.degree. C.
[0201] 3--The lysat is processed for total nucleic acids
purification with funnel-design having silica membrane in mini prep
spin columns able to concentrate nucleic acid. The elution volume
is up to 100 .mu.L. 2 .mu.L (up to 5 .mu.L) of pure nucleic acids
extract is used for one step RT-PCRs with enzyme like Tth and for
one step PCRs with enzyme like DNA Polymerase. The following
program is peformed for the one step RT-PCR: I: Reverse
transcription 48.degree. C./15 min II: Denaturation 95.degree.
C./10 minutes III: PCR (40 cycles) 95.degree. C./15 seconds,
60.degree. C./60 seconds. The following program is peformed for the
one step PCR: I: Denaturation 95.degree. C./30 seconds III: PCR (45
cycles) 95.degree. C./10 seconds, 60.degree. C./10 seconds,
72.degree. C./35 seconds.
[0202] One step PCRs, using the primers SEQ ID No 1, SEQ ID No 2,
SEQ ID No 7, SEQ ID No 8, SEQ ID No 9 and SEQ IDNo 10 allow to
amplified the DNA from Prevotella melaminogenica, Streptococcus
mitis and Capnocytophaga gingivalis respectively. The detection and
quantification system is a Syber Green system or a probe system
using the SEQ ID No 32 or SEQ ID No 49 for Prevotella
melaminogenica quantification, SEQ ID No 33 or SEQ ID No 51 for
Streptococcus mitis quantification and SEQ ID No No 34 or SEQ ID No
52 for Capnocytophaga gingivalis quantification.
[0203] One step RT-PCRs, using the primers SEQ ID No 26, SEQ ID No
27 or SEQ ID No 28, SEQ ID No 29 and SEQ ID No 22 and SEQ ID No 23
allow to amplify H3F3A and SSAT mRNA respectively. The detection
and quantification system could a Syber Green system or a probe
system using the SEQ ID No 44 or SEQ ID No 58 or SEQ ID No 45 or
SEQ ID No 46 or SEQ ID No 59 for H3F3A quantification and SEQ ID No
42 or SEQ ID No 57 for SSAT quantification. Each assay is confirmed
using commercial probe kits (ABI) that amplified the H3F3A gene
NM005324 on the exon 4 and the SSAT gene NM 002970 on the exons 3
and 4.
[0204] The results obtained for the patients from the oral cancer
population versus the healthy individuals are presented in table
1.
TABLE-US-00001 TABLE 1 overexpression of genetic biomarker in oral
cancer population Overexpression Genetic biomarker in oral cancer
name population Streptococcus mitis 2.80 X Capnocytophaga 2.00 X
gingivalis Prevotella 2.30 X melaninogenica SSAT 2.63 X H3F3A 2.56
X
[0205] It has been concluded from these data that the "detection"
of a certain genetic marker (DNA or RNA) in a saliva sample of a
patient means that said sample contains at least 2 fold the amount
of said marker in the normal population.
[0206] Importantly, the tumorigenic status of 11 samples among 17
has been detected using the genetic markers SSAT, of H3F3A and the
sequence of the bacteria Streptococcus mitis. Therefore, it can be
conclude that the detection of these at least three genetic marker
in the saliva of a human subject enables to diagnose an oral cancer
with a sensibility of 64%. In other word, the detection of the mRNA
of SSAT, the mRNA of H3F3A and the bacteria sequence of
Streptococcus mitis indicates that the human subject has a risk
superior to 60% of developping an oral cancer.
[0207] Accordingly, IL8 is not considered to be a significant
marker for oral cancer in saliva.
Example 7
Analysis of Organic Volatile Compounds in the Volatile Fraction of
Saliva
[0208] It is known for a while that volatile compounds can be
extracted from fluidic samples from oral cavity giving the
possibility to explore the saliva as material to be analyzed for
pathogenic diseases (Volozhin et al. Stomatologiia (mosk), 2001;
80(1):9-12).
[0209] In the present case, 1 ml of saliva solution is placed in a
glass vial with 10 .mu.L of the standard solution with 1 ppm of
three (3) standards (1-bromobutane, 1-bromobenzene and
1.4-dibromobenzene; final solution with 1 ppm prepared in pure
water).
[0210] The samples are placed at room temperature during at least 1
hour before analyzes. The sample is heated at 40.degree. C. during
10 minutes then the extraction of the volatile compounds is carried
out at 40.degree. C., using a CAR/PDMS fiber (SPME fiber assembly
CAR/PDMS of 75 .mu.m (Supelco, Bellefont, Pa., USA)), during 30
minutes. Then the analysis was performed using GC/MS. The GC
injection port temperature is 280.degree. C. The injection of the
volatile molecules in GC/MS is carried out by thermal desorption of
the fiber at 280.degree. C. The separation of the volatile
compounds was led with a non-polar capillary column. The column
temperature program was: initial temperature of 40.degree. C. for 5
min, then increase at 3.degree. C./min to 230.degree. C. for 2 min.
The mass spectra are measured by electronic impact at 70 e.V.
[0211] The identification of the volatile molecules is obtained by:
[0212] comparison of the experimental indices of retention to those
of the internal data bank [0213] comparison of the experimental
spectra to those of the bank Wiley 275K.
[0214] The results of the exhaustive analysis of all the volatile
organic compounds found in the volatile fraction of human saliva
are reported on table 2.
TABLE-US-00002 TABLE 2 Organic volatile compounds in volatile
fraction of human saliva Molecule name CAS number
1,2-dichlorobenzene 95-50-1 disulfure de carbone 137-26-8
benzenecarboxylic acid 65-85-0 heptanoic acid 111-14-8 nonanoic
acid 112-05-0 octanoic acid 124-07-2 pentanoic acid 109-52-4
2-propenal, 2-methyl- 78-85-3 butanal 123-72-8 butanal, 3-methyl-
590-86-3 decanal 112-31-2 ethanal 75-07-0 heptanal 111-71-7 hexanal
66-25-1 hexanal, 2-ethyl- 123-05-7 nonanal 124-19-6 octanal
124-13-0 propanal, 2-methyl- 78-84-2 valeraldehyde,
4,4-dimethyl-2-methylene 5375-28-0 7-oxabicyclo[4.1.0]heptane,
1-methyl- 1713-33-3 cyclopentane, methyl- 96-37-7 decane 124-18-5
hexane 110-54-3 methylcyclohexane 108-87-2 nonane 111-84-2 pentane,
2,2,4-trimethyl- 540-84-1 pentane, 2-methyl- 107-83-5 pentane,
3-methyl- 96-14-0 benzene 71-43-2 benzene, ethyl- 100-41-4
Butylated Hydroxytoluene 128-37-0 dehydro p-cymene 1195-32-0
dibenzofuran 132-64-9 m-xylene 108-38-3 o-xylene 95-47-6 p-cymene
99-87-6 styrene 100-42-5 toluene 108-88-3 1-hexene,
3,5,5-trimethyl- 4316-65-8 2-hexene, 2,5,5-trimethyl- 40467-04-7
butanoic acid, 2-methyl-, ethyl ester 7452-79-1 butanoic acid,
3-methyl-, ethyl ester 108-64-5 methyl thiolacetate 1534-08-3 ethyl
butanoate 105-54-4 ethyl propanoate 105-37-3
ethyl-N-methylcarbamate 105-40-8 methylacetate 79-20-9
1,3-dioxolane, 2-methyl- 497-26-7 tert-butyl ethyl ether 637-92-3
bromoethane 74-96-4 bromomethane 74-83-9 chlorobutanol 57-15-8
dibromomethane 74-95-3 dichloromethane 75-09-2 heptane, 3-bromo-
1974-05-6 hexane, 1-chloro- 544-10-5 hexane, 3-chloro- 2346-81-8
phenol, 2-chloro-4-(1,1-dimethylpropyl)- 98-28-2 tribromomethane
75-25-2 1H-pyrrole 109-97-7 1H-pyrrole, 1-methyl- 96-54-8
2-furfural 98-01-1 furan, 2-acetyl- 1192-62-7 furan, 2-ethyl-
3208-16-0 furan, 2-pentyl- 3777-69-3 furan, 3-methyl 930-27-8
furan, 5-methyl-2-propionyl- 33978-70-0 pyrazine 290-37-9 pyrazine,
2,5-dimethyl- 123-32-0 pyrazine, ethyl- 13925-00-3 pyrazine,
methyl- 109-08-0 pyridine 110-86-1 2-butenenitrile 4786-20-3
2-piperidinone 675-20-7 3-butenenitrile 109-75-1 benzene isocyanato
103-71-9 benzonitrile 100-47-0 butanenitrile, 3-methyl- 625-28-5
ethane, isocyano- 624-79-3 ethyl isocyanate 109-90-0 propanenitrile
107-12-0 propanenitrile, 2-methyl- 78-82-0 1-hexanol 111-27-3
1-hexanol, 2-ethyl- 104-76-7 1-pentanol 71-41-0 1-propanol,
2-methyl- 78-83-1 2-butanol 15892-23-6 2-butanol, 2-methyl- 75-85-4
2-hexanol, 2,5-dimethyl- 3730-60-7 2-pentanol, 2-methyl- 590-36-3
2-propanol, 2-methyl- 75-65-0 3-hexanol 623-37-0 ethanol 64-17-5
phenol 108-95-2 phenol, 4-(1,1-dimethylpropyl)- 80-46-6 skatole
83-34-1 1,3-isobenzofuranedione 85-44-9 2,3-octanedione 585-25-1
2,4-pentanedione, 3-methyl- 815-57-6
2,6-di-tert-butyl-p-benzoquinone 719-22-2 2-butanone 78-93-3
2-butanone, 3,3-dimethyl- 75-97-8 2-butanone, 3-hydroxy- 513-86-0
2-cyclohexen-1-one, 3,4,4-trimethyl- 17299-41-1
2-cyclopenten-1-one, 2,3-dimethyl- 1121-05-7 2-cyclopenten-1-one,
3-methyl- 2758-18-1 2-hexanone, 3-methyl- 2550-21-2
2-methyl-2-cyclopenten-1-one 1120-73-6 3-heptanone 106-35-4
3-hepten-2-one, 5-methyl- 5090-16-4 3-octanone 106-68-3
3-penten-2-one, 3-methyl- 565-62-8 5-hepten-2-one, 6-methyl-
110-93-0 acetophenone 98-86-2 cyclohexanone 108-94-1
cyclopentanone, 2-methyl- 1120-72-5 cyclopentanone, 3-methyl-
1757-42-2 methylbutanone 563-80-4 p-methylacetophenone 122-00-9
disulfide, dimethyl 624-92-0 methanethiol 74-93-1 sulfone,
dimethyl- 67-71-0 dimethyl sulfide 75-18-3 (Z)-caryophyllene
118-45-0 anethole (E) 4180-23-8 a-pinene 80-56-8 a-terpineol
10482-56-1 a-thujene 2867-05-2 b-bourbonene 5208-59-3
b-caryophyllene 87-44-5 b-pinene 127-91-3 camphene 79-92-5 carvone
2244-16-8 cis-p-menthan-3-one 491-07-6 dihydromyrcenol 18479-59-9
eucalyptol 470-82-6 limonene 138-86-3 m-mentha-6,8-diene 1461-27-4
neo-menthol 491-01-0 piperitone 89-81-6 p-menth-3-ene 500-00-5
trans-p-menthan-3-one 89-80-5 benzaldehyde 100-52-7 2-octanone
111-13-7 2-heptanone 110-43-0 2,3-pentanedione 600-14-6
2-pentanone, 3-methyl- 565-61-7 acetic acid 64-19-7 3-hexanone
589-38-8 1-propene, 2-methyl- 115-11-7 benzaldehyde, 2-methyl-
529-20-4 propanoic acid 79-09-4 butanoic acid 107-92-7 3-pentanone,
2-methyl- 565-69-5 2-pentanone 4,4-dimethyl- 590-50-1 ethyl acetate
141-78-6 thiophene, 3-methyl- 616-44-4 2-pentanone 107-87-9
2-hexanone 591-78-6 isovaleric acid 503-74-2 2-pentanone,4-methyl-
108-10-1 2-butene, 2-methyl- 513-35-9 1-propanol 71-23-8 2-butenal,
2-methyl (E) 497-03-0 butanoic acid, 2-methyl- 116-53-0 2-propanol
67-63-0 isobutyric acid 79-31-2 benzene, 2-methyl-1-propenyl-
768-49-0 hexanoic acid 142-62-1 acetone 67-64-1 pentanoic acid,
4-methyl- 646-07-1 cyclopropane, 1,2-dimethyl (cis) 930-18-7
p-cresol 106-44-5 2,3-butanedione 431-03-8 1-butanol, 3-methyl-
123-51-3 m-methyl acetophenone 585-74-0 3-hexen-2-one, 5-methyl-
5166-53-0 indole 120-72-9 3-hexen-2-one, 3,4-dimethyl- 1635-02-5
pentanal 110-62-3 2-pentanol, 2,3-dimethyl- 4911-70-0 m-cymene
535-77-3 cyclohexanone 108-94-1 aniline 62-53-3 furan, 2-methyl-
534-22-5 3-pentanone, 2,4 dimethyl- 565-80-0 3-buten-2-one 3-methyl
814-78-8 hexanenitrile 628-73-9 heptanenitrile 629-08-3 pentane
nitrile 110-59-8 butanenitrile 109-74-0 acrylonitrile 107-13-1
[0215] From our experimental studies, 192 volatile molecules have
been identified in the volatile fraction of human saliva (table 2).
Principal volatile compounds identified in saliva are ketones,
acids, aldehydes, alcohols and aromatic compounds.
[0216] Among these compounds, 57 volatile compounds have been
preselected to be used has possible biomarkers discriminating
factor for oral cancer early detection (table 3).
TABLE-US-00003 TABLE 3 Volatile compounds potentially indicative of
oral cancer susceptibility Molecule name CAS Number benzaldehyde
100-52-7 2-octanone 111-13-7 2-heptanone 110-43-0 2,3-pentanedione
600-14-6 3-methyl-2-pentanone 565-61-7 acetic acid 64-19-7
3-hexanone 589-38-8 2-methyl-1-propene 115-11-7
2-methyl-benzaldehyde 529-20-4 propanoic acid 79-09-4 butanoic acid
107-92-7 2-methyl-3-pentanone 565-69-5 4,4-dimethyl-2-pentanone
590-50-1 ethyl acetate 141-78-6 3-methyl-thiophene 616-44-4
2-pentanone 107-87-9 2-hexanone 591-78-6 isovaleric acid 503-74-2
4-methyl-2-pentanone 108-10-1 2-methyl-2-butene 513-35-9 1-propanol
71-23-8 (E) 2-methyl-2-butenal 497-03-0 2-methyl-butanoic acid
116-53-0 2-propanol 67-63-0 isobutyric acid 79-31-2
2-methyl-1-propenyl-benzene 768-49-0 hexanoic acid 142-62-1 acetone
67-64-1 4-methyl-pentanoic acid 646-07-1 (cis) 1,2-dimethyl
cyclopropane 930-18-7 p-cresol 106-44-5 2,3-butanedione 431-03-8
3-methyl-1-butanol 123-51-3 m-methyl acetophenone 585-74-0
5-methyl-3-hexen-2-one 5166-53-0 indole 120-72-9
3,4-dimethyl-3-hexen-2-one 1635-02-5 pentanal 110-62-3
2,3-dimethyl-2-pentanol 4911-70-0 m-cymene 535-77-3 cyclohexanone
108-94-1 aniline 62-53-3 2-methyl-furan 534-22-5 2,4
dimethyl-3-pentanone 565-80-0 3-methyl-3-buten-2-one 814-78-8
hexanenitrile 628-73-9 heptanenitrile 629-08-3 pentane nitrile
110-59-8 butanenitrile 109-74-0 3-methyl-2-pentanone 565-61-7
methylbutanone 563-80-4 butanal 123-72-8 hexanal 66-25-1 phenol
108-95-2 m-xylene 108-38-3 p-xylene 106-42-3 ethanal 75-07-0
benzene 71-43-2 acrylonitrile 107-13-1
[0217] According to our results, at least 19 of these 57 compounds
are indeed correlated with oral cancer, as shown below.
Example 8
Quantification of the Identified Volatile Compounds in
Cancer/Healthy Patients
[0218] The quantification of the volatile compounds is made by
comparison with standard controls that have been added in the
preservation buffer at the beginning of the experiment. In this
case, the followings molecular standards have been used: [0219]
1-bromobutane (CAS number 109-65-9) [0220] 1-bromobenzene (CAS
number 108-86-1) [0221] 1,4-dibromobenzene (CAS number
106-37-6)
TABLE-US-00004 [0221] TABLE 4 Particular biomarkers indicative of
oral cancer predisposition Overexpression in oral cancer
Overexpression in Biomarker name population normal population
2,3-pentanedione 6.00 X 3-methyltiophene 1.50 X acetone 2.30 X
hexanitrile 3.00 X benzaldehyde 1.80 X 3-methyl-2- 2.10 X pentanone
2,3-butanedione 4.40 X 2-propanol 2.80 X ethyl acetate 3.90 X
1-propanol 1.90 X hexanal 1.60 X 5-methyl-3-hexen- 1.70 X 2-one m-
and p-xylene 1.50 X 2-methyl-2-butenal 1.90 X (E) 2-methyl-2-butene
2.00 X 2-methyl-1- 3.30 X propene (cis) 1,2-dimethyl 1.70 X
cyclopropane
[0222] It has been concluded from these data that the "detection"
of a certain volatile compound in a saliva sample of a patient
means that said sample contains at least 1.5 fold the amount of
said compound in the normal population.
Example 9
Statistical Analysis of the Presence of Biochemical Organic
Compounds in the Saliva of Oral Cancer Patient Vs Healthy
Individuals
[0223] Software STATISTICA version 8.0 of StatSoft France (2007) is
used for data analysis. The significances of the differences
between the groups were tested by from Factorial Discriminating
Analysis (FDA). Thus the similarities or the differences of the
samples can be visualized graphically.
[0224] The identification of the volatile molecules is obtained by:
[0225] Comparison of the experimental indices of retention to those
of the internal data bank, [0226] comparison of the experimental
spectra to those of the bank Wiley 275K and NIST 2.0d, built april
2005. [0227] Statistical model 1
[0228] Total population tested is 45 human subjects from two
distinctive environmental geographic areas. Oral cancer population
is confirmed by visual diagnostics performed by an anticancer
center.
[0229] The statistical analyzes were carried out on 109 volatile
compounds. Abundances of the molecules in each sample were reported
to abundances of the 3 internal standards analyzed with saliva. The
principal volatile compounds identified in saliva are ketones,
acids, aldehydes, alcohols and aromatic compounds. All the samples
have a strong abundance in hydrazoic acid coming directly from the
buffer solution of conservation.
[0230] On the 108 volatile compounds, 49 are significant to
separate the group "tumor" from the reference group. A
discriminating factorial analysis on these variables makes it
possible to classify well 97.78% of the samples with 4 volatile
compounds: the hexanenitrile, the 2,3-pentanedione,
3-methylthiophene and acetone. Only 1 false-positive have been
detected with the statistical model 1. [0231] Statistical model
2
[0232] Total population tested is 45 human subjects from two
distinctive environmental geographic areas. Oral cancer population
is confirmed by visual diagnostics performed by a specialized
anticancer center.
[0233] The statistical analyzes were carried out on 108 volatile
compounds. Abundances of the molecules in each sample were reported
to abundances of the 3 internal standards analyzed with saliva. The
principal volatile compounds identified in saliva are ketones,
acids, aldehydes, alcohols and aromatic compounds. All the samples
have a strong abundance in hydrazoic acid coming directly from the
buffer solution of conservation.
[0234] All nitriles volatile compounds have been removed from the
statistical model number 2. For this study, the 10 made up
"nitriles" were not taken into account.
[0235] From the 98 remaining volatile compounds, an ANOVA test
according to the factor "tumor" showed that 45 components are
significant to separate the group "tumor" from the reference
group.
[0236] A discriminating factorial analysis on these variables makes
it possible to classify well 93.33% of the samples with 4 volatile
compounds: benzaldehyde, acetone, the 2,3-pentanedione and
2-methyl-2-butene. The first 3 molecules are side of the group
"tumor" and the 2-methyl-2-butene on the side of the control group.
3 false-negatives and no false positive have been detected with the
statistical model 2.
[0237] To conclude, this study highlights the tight link existing
between 14 organic compounds (namely hexanenitrile, the
2,3-pentanedione, 3-methylthiophene, 2-methyl-2-butylene,
3-methyl-2-pentanone, 2,3-butanedione, 2-propanol, ethyl acetate,
1-propanol, hexanal, 5-methyl-3-hexen-2-one, m-xylene, p-xylene,
2-methyl-2-butenal (E)) and oral cancer in human. It is noteworthy
that none of them have ever been found in exhaled breath (Mashir A,
Advanced Powder Technology, 2009) or being associated to oral
cancer.
Example 10
Diagnostic Test Based on the Ratios of Specific Organic
Molecules
[0238] Software STATISTICA version 8.0 of StatSoft France (2007) is
used for data analysis. The significances of the differences
between the groups were tested by Factorial Discriminating Analysis
(FDA). Thus the similarities or the differences of the samples can
be visualized graphically.
Tested Population
[0239] Total population tested is 52 human subjects from two
distinctive environmental geographic areas. Oral cancer population
is confirmed by visual diagnostics performed by a specialized
anticancer center.
[0240] The following volatile organic compounds are used in the
diagnostic test: [0241] 3-methyl-2-pentanone (CAS number: 565-61-7)
[0242] Methylbutanone (CAS number: 563-80-4) [0243]
2.4-dimethyl-3-pentanone (CAS number: 565-80-0) [0244] Benzene (CAS
number: 71-43-2) [0245] Phenol (CAS number: 108-95-2) [0246]
2,3-butanedione (CAS number: 431-03-8) [0247]
5-methyl-3-hexen-2-one (CAS number: 5166-53-0) [0248]
2-methyl-1-propene (CAS number: 115-11-7) [0249] Butanal (CAS
number: 123-72-8) [0250] Hexanal (CAS number: 66-25-1) [0251]
2-propanol (CAS number: 67-63-0) [0252] Ethyl acetate (CAS number:
141-78-6) [0253] Hexanenitrile (CAS number: 628-73-9) [0254]
1-propanol (CAS number: 71-23-8) [0255] (cis)
1,2-dimethyl-cyclopropane (CAS number: 930-18-7) [0256] m- and
p-xylene (CAS number: 108-38-3 and CAS number: 106-42-3) [0257] (E)
2-methyl-2-butenal (CAS number: 497-03-0) [0258] 3-methyl-thiophene
(CAS number: 616-44-4) [0259] Ethanal (CAS number: 75-07-0)
[0260] The median values by group were calculated for the following
ratios: [0261] 3-methyl-2-pentanone/methyl butanone [0262]
2,4-dimethyl-3-pentanone/benzene [0263] phenol/2,3-butanedione
[0264] 5-methyl-3-hexen-2-one/2-methyl-1-propene [0265]
butanal/hexanal [0266] 2-propanol/ethyl acetate [0267]
hexanenitrile/1-propanol [0268] 2-propanol/(cis) 1.2-dimethyl
cyclopropane [0269] m-xylene/2-methyl-2-butenal [0270]
3-methyl-2-pentanone/3-methyl-thiophene, [0271]
2,3-butanedione/ethanal
[0272] The statistical method used is FDA (Factorial Discrimination
Analysis).
[0273] The median values by group were calculated for each of the
ratios.
[0274] The 5 followings were found to be statistically
significative of oral cancer patient group or control group: [0275]
1) 3-methyl-2-pentanone/methyl butanone (R1) [0276] 2)
Butanal/hexanal (R56) [0277] 3) Hexanenitrile/1-propanol (R260)
[0278] 4) 2-propanol/(cis) 1,2 dimethyl cyclopropane (R266) [0279]
5) Phenol/2,3 butanedione (R269).
[0280] Among these ratios, 2 were found to be reproducibly
correlated with healthy subjects, and three were indicative of oral
cancer suffering patients (table 5).
TABLE-US-00005 TABLE 5 Median values for the 5 ratios correlated
with oral cancer or healthiness R266 R1 2-propanol/ 3-methyl-2- R56
R260 (cis) 1,2- R269 pentanone/ Butanal/ Hexanenitrile/ dimethyl
Phenol/2,3- methyl butanone hexanal 1-propanol cyclopropane
butanedione Average 0.182 0.223 0.040 2.939 0.696 healthy Average
0.357 0.138 0.089 9.359 0.217 Oral Cancer
[0281] The 3 ratios R1 (3-methyl-2-pentanone/methyl butanone), 8260
(Hexanenitrile/1-propanol) and R266(Phenol/2,3-butanedione) are of
the side of the oral cancer group group and the values are
respectively 1.96; 2.24 and 3.18 times higher in this group than in
the healthy group.
[0282] The 2 ratios R56 (Butanal/hexanal) and 8269
(Phenol/2,3-butanedione) are of the side of the healthy group and
are respectively 1.62 and 3.21 higher in this group than in the
oral cancer group.
[0283] The absolute limiting values of the ratios permitting to
classify the patients in a potential "oral cancer group" are given
in table 6:
TABLE-US-00006 TABLE 6 Absolute limiting ratio values permitting to
classify the patients Condition so that the Nb samples sample is
"oral cancer corresponding to Report/ratio risk" the ratio
3-methyl-2-pentanone/methyl >0.344 17 butanone Butanal/hexanal
<0.11 10 Hexanenitrile/1-propanol >0.167 3 2-propanol/(cis)
1,2-dimethyl >10.33 9 cyclopropane Phenol/2,3-butanedione
<0.005 4
[0284] To classify the samples it is necessary to apply the
following formula (from the FDA statistical method) taking into
account all these 5 ratios (linear combination of the 5 variable
ratios):
Factor
X=1.8277-7.3472*R1-0.125*R266-14.2293*R260+1.2050*R269+8.883*R56
[0285] If factor X.ltoreq.0.6, the sample is classified in the Oral
Cancer Risk Population
[0286] If factor X>0.6, the sample is classified healthy
[0287] Therefore, the method of the invention, based on: [0288] i)
the recovery of the volatile fraction of the saliva of a human
subject, [0289] i) the quantification of ten biochemical organic
compounds (3-methyl-2-pentanone, methyl butanone, Butanal, hexanal,
Hexanenitrile, 1-propanol 2-propanol, (cis) 1,2-dimethyl
cyclopropane, phenol, and 2,3-butanedione) in said volatile
fraction, [0290] ii) calculation of the ratios R1, R266, 8260, 8269
and R56 as mentioned above, [0291] iii) calculation of said factor
X and its comparison with the threshold 0.6, enables the man
skilled in the art to prognose and/or diagnose an oral cancer in
said human subject.
[0292] The analysis of the ratios between the organic compounds:
3-methyl-2-pentanone/methyl butanone, Butanal/hexanal,
Hexanenitrile/1-propanol 2-propanol/(cis) 1,2-dimethyl
cyclopropane, and Phenol/2,3-butanedione in the volatile fraction
of the saliva of a human subject permits to obtain a highly
sensitive test of predisposition of oral cancer (98.077%
sensitivity; 1 false-positive) (FIG. 1).
Example 11
Combination of the Biomarkers of Fluid Fraction and Volatile
Fraction for Diagnosing/Prognosing Oral Cancer
[0293] Software STATISTICA version 8.0 of StatSoft France (2007) is
used for data analysis. The significances of the differences
between the groups were obtained from Factorial Discriminating
Analysis (FDA). Thus the similarities or the differences of the
samples can be visualized graphically.
[0294] The best combination of biomarker was the following:
Streptococcus mitis+SSAT+H3F3A+hexanenitrile+2.3-pentanedione,
3-methylthiophene+acetone. Indeed the use of such markers permits
to obtain 100% sensibility results as shown in table 7.
TABLE-US-00007 TABLE 7 Sensibility of the diagnostic test based on
biomarkers from the fluid fraction and volatile fraction volatile
bac- bacteria + compounds teria + hmRNA + human statistical human
volatile bacteria mRNA model 1 mRNA compounds Specificity % 76 54
98 83 98 Sensibility % 79 32 100 89 100
Sequence CWU 1
1
70120DNAartificial sequencePrimer forward 1gtgggataac ctgccgaaag
20225DNAartificial sequencePrimer reverse 2cccatccatt accgataaat
cttta 25321DNAartificial sequencePrimer forward 3ggcagactaa
tacctgcata g 21424DNAartificial sequencePrimer reverse 4atccattacc
gataaacttt cttc 24525DNAartificial sequencePrimer reverse
5catccccatc cattaccgat aaatc 25621DNAartificial sequencePrimer
forward 6taccgcataa gagtagatgt t 21721DNAartificial sequencePrimer
forward 7cgatagctaa taccgcataa g 21821DNAartificial sequencePrimer
reverse 8caggtccatc tggtagtgat g 21923DNAartificial sequencePrimer
forward 9ggatagcccg gagaaatttg gat 231027DNAartificial
sequencePrimer reverse 10cgtcatcaaa gtacacgtac tccttat
271121DNAartificial sequencePrimer forward 11taccggatag gagcgtccac
c 211221DNAartificial sequencePrimer reverse 12taggccgcga
gtccatccaa a 211325DNAartificial sequencePrimer forward
13ttgcagatca tcaagaacac gtaga 251427DNAartificial sequencePrimer
reverse 14cagtagagat cagttgtctc tggttgc 271519DNAartificial
sequencePrimer forward 15agaggccagt gccattcgt 191621DNAartificial
sequencePrimer reverse 16gtttctctgc gtcgttggag t
211718DNAartificial sequencePrimer forward 17cgtcttcccc tccatcgt
181823DNAartificial sequencePrimer reverse 18agctcattgt agaaggtgtg
gtg 231921DNAartificial sequencePrimer forward 19aagacctgta
cgccaacaca g 212020DNAartificial sequencePrimer reverse
20cgtcatactc ctgcttgctg 202122DNAartificial sequencePrimer reverse
21atactcctgc ttgctgatcc ac 222221DNAartificial sequencePrimer
forward 22ggatcagaaa ttctgaagaa t 212321DNAartificial
sequencePrimer reverse 23accctcttca ctggacagat c
212421DNAartificial sequencePrimer forward 24ccagtgaaga gggttggaga
c 212522DNAartificial sequencePrimer reverse 25tggaggttgt
catctacagc ag 222620DNAartificial sequencePrimer forward
26ggcgctccgt gaaattagac 202719DNAartificial sequencePrimer reverse
27cgctggaagg gaagtttgc 192819DNAartificial sequencePrimer forward
28aaagcaccca ggaagcaac 192922DNAartificial sequencePrimer reverse
29gcgaatcaga agttcagtgg ac 223022DNAartificial sequencePrimer
forward 30gagggttgtg gagaagtttt tg 223122DNAartificial
sequencePrimer reverse 31ctggcatctt cactgattct tg
223230DNAartificial sequenceProbe forward 32gcatagtctt cgatgacggc
atcagatttg 303321DNAartificial sequenceProbe forward 33gatgttccat
gacatttrct t 213421DNAartificial sequenceProbe forward 34attggatggc
atcatttgat a 213518DNAartificial sequenceProbe forward 35catggtgggt
gttggaaa 183633DNAartificial sequenceProbe forward 36aatcatgcat
ggagatacac ctacattgca tga 333722DNAartificial sequenceProbe forward
37gctgcaaccg agcacgacag ga 223818DNAartificial sequenceProbe
forward 38caggcaccag ggcgtgat 183916DNAartificial sequenceProbe
forward 39cgagcacggc atcgtc 164018DNAartificial sequenceProbe
forward 40gcagatgtgg atcagcaa 184122DNAartificial sequenceProbe
forward 41aggatgcaga aggagatcac tg 224221DNAartificial
sequenceProbe forward 42taagccaggt tgcaatgagg t 214321DNAartificial
sequenceProbe forward 43gcactcctca ctcctctgtt g 214426DNAartificial
sequenceProbe forward 44tatcagaagt ccactgaact tctgat
264529DNAartificial sequenceProbe forward 45atcagaagtc cactgaactt
ctgatycgc 294620DNAartificial sequenceProbe forward 46ggcgctccgt
gaaattagac 204719DNAartificial sequenceProbe forward 47ttcattctct
gtggtatcc 194817DNAartificial sequenceProbe forward 48ttcattctct
gtggtat 174930DNAartificial sequenceProbe reverse 49caaatctgat
gccgtcatcg aagactatgc 305021DNAartificial sequenceProbe reverse
50aagyaaatgt catggaacat c 215121DNAartificial sequenceProbe reverse
51aagyaaatgt catggaacat c 215221DNAartificial sequenceProbe reverse
52tatcaaatga tgccatccaa t 215318DNAartificial sequenceProbe reverse
53tttccaacac ccaccatg 185418DNAartificial sequenceProbe reverse
54atcacgccct ggtgcctg 185516DNAartificial sequenceProbe reverse
55gacgatgccg tgctcg 165622DNAartificial sequenceProbe reverse
56cagtgatctc cttctgcatc ct 225721DNAartificial sequenceProbe
reverse 57acctcattgc aacctggctt a 215826DNAartificial sequenceProbe
reverse 58atcagaagtt cagtggactt ctgata 265920DNAartificial
sequenceProbe reverse 59gtctaatttc acggagcgcc 206017DNAartificial
sequenceProbe reverse 60ataccacaga gaatgaa 17611761DNAHomo
sapiensmisc_featureHuman mRNA for beta-actin - NCBI X00351
61ttgccgatcc gccgcccgtc cacacccgcc gccagctcac catggatgat gatatcgccg
60cgctcgtcgt cgacaacggc tccggcatgt gcaaggccgg cttcgcgggc gacgatgccc
120cccgggccgt cttcccctcc atcgtggggc gccccaggca ccagggcgtg
atggtgggca 180tgggtcagaa ggattcctat gtgggcgacg aggcccagag
caagagaggc atcctcaccc 240tgaagtaccc catcgagcac ggcatcgtca
ccaactggga cgacatggag aaaatctggc 300accacacctt ctacaatgag
ctgcgtgtgg ctcccgagga gcaccccgtg ctgctgaccg 360aggcccccct
gaaccccaag gccaaccgcg agaagatgac ccagatcatg tttgagacct
420tcaacacccc agccatgtac gttgctatcc aggctgtgct atccctgtac
gcctctggcc 480gtaccactgg catcgtgatg gactccggtg acggggtcac
ccacactgtg cccatctacg 540aggggtatgc cctcccccat gccatcctgc
gtctggacct ggctggccgg gacctgactg 600actacctcat gaagatcctc
accgagcgcg gctacagctt caccaccacg gccgagcggg 660aaatcgtgcg
tgacattaag gagaagctgt gctacgtcgc cctggacttc gagcaagaga
720tggccacggc tgcttccagc tcctccctgg agaagagcta cgagctgcct
gacggccagg 780tcatcaccat tggcaatgag cggttccgct gccctgaggc
actcttccag ccttccttcc 840tgggcatgga gtcctgtggc atccacgaaa
ctaccttcaa ctccatcatg aagtgtgacg 900tggacatccg caaagacctg
tacgccaaca cagtgctgtc tggcggcacc accatgtacc 960ctggcattgc
cgacaggatg cagaaggaga tcactgccct ggcacccagc acaatgaaga
1020tcaagatcat tgctcctcct gagcgcaagt actccgtgtg gatcggcggc
tccatcctgg 1080cctcgctgtc caccttccag cagatgtgga tcagcaagca
ggagtatgac gagtccggcc 1140cctccatcgt ccaccgcaaa tgcttctagg
cggactatga cttagttgcg ttacaccctt 1200tcttgacaaa acctaacttg
cgcagaaaac aagatgagat tggcatggct ttatttgttt 1260tttttgtttt
gttttggttt tttttttttt tttggcttga ctcaggattt aaaaactgga
1320acggtgaagg tgacagcagt cggttggagc gagcatcccc caaagttcac
aatgtggccg 1380aggactttga ttgcacattg ttgttttttt aatagtcatt
ccaaatatga gatgcattgt 1440tacaggaagt cccttgccat cctaaaagcc
accccacttc tctctaagga gaatggccca 1500gtcctctccc aagtccacac
aggggaggtg atagcattgc tttcgtgtaa attatgtaat 1560gcaaaatttt
tttaatcttc gccttaatac ttttttattt tgttttattt tgaatgatga
1620gccttcgtgc ccccccttcc ccctttttgt cccccaactt gagatgtatg
aaggcttttg 1680gtctccctgg gagtgggtgg aggcagccag ggcttacctg
tacactgact tgagaccagt 1740tgaataaaag tgcacacctt a 1761621060DNAHomo
sapiensmisc_featureHomo sapiens spermidine/spermine
N1-acetyltransferase 1 (SAT1), mRNA NCBI NM_002970 62cgcgggccga
ctggtgttta tccgtcactc gccgaggttc cttgggtcat ggtgccagcc 60tgactgagaa
gaggacgctc ccgggagacg aatgaggaac cacctcctcc tactgttcaa
120gtacaggggc ctggtccgca aagggaagaa aagcaaaaga cgaaaatggc
taaattcgtg 180atccgcccag ccactgccgc cgactgcagt gacatactgc
ggctgatcaa ggagctggct 240aaatatgaat acatggaaga acaagtaatc
ttaactgaaa aagatctgct agaagatggt 300tttggagagc acccctttta
ccactgcctg gttgcagaag tgccgaaaga gcactggact 360ccggaaggac
acagcattgt tggttttgcc atgtactatt ttacctatga cccgtggatt
420ggcaagttat tgtatcttga ggacttcttc gtgatgagtg attatagagg
ctttggcata 480ggatcagaaa ttctgaagaa tctaagccag gttgcaatga
ggtgtcgctg cagcagcatg 540cacttcttgg tagcagaatg gaatgaacca
tccatcaact tctataaaag aagaggtgct 600tctgatctgt ccagtgaaga
gggttggaga ctgttcaaga tcgacaagga gtacttgcta 660aaaatggcaa
cagaggagtg aggagtgctg ctgtagatga caacctccat tctattttag
720aataaattcc caacttctct tgctttctat gctgtttgta gtgaaataat
agaatgagca 780cccattccaa agctttatta ccagtggcgt tgttgcatgt
ttgaaatgag gtctgtttaa 840agtggcaatc tcagatgcag tttggagagt
cagatctttc tccttgaata tctttcgata 900aacaacaagg tggtgtgatc
ttaatatatt tgaaaaaaac ttcattctcg tgagtcattt 960aaatgtgtac
aatgtacaca ctggtactta gagtttctgt ttgattcttt tttaataaac
1020tactctttga tttaaaaaaa aaaaaaaaaa aaaaaaaaaa 1060631117DNAHomo
sapiensmisc_featureHomo sapiens H3 histone, family 3A (H3F3A), mRNA
NCBI NM_002107 63caattgtgtt cgcagccgcc gccgcgccgc cgtcgctctc
caacgccagc gccgcctctc 60gctcgccgag ctccagccga aggagaaggg gggtaagtaa
ggaggtctct gtaccatggc 120tcgtacaaag cagactgccc gcaaatcgac
cggtggtaaa gcacccagga agcaactggc 180tacaaaagcc gctcgcaaga
gtgcgccctc tactggaggg gtgaagaaac ctcatcgtta 240caggcctggt
actgtggcgc tccgtgaaat tagacgttat cagaagtcca ctgaacttct
300gattcgcaaa cttcccttcc agcgtctggt gcgagaaatt gctcaggact
ttaaaacaga 360tctgcgcttc cagagcgcag ctatcggtgc tttgcaggag
gcaagtgagg cctatctggt 420tggccttttt gaagacacca acctgtgtgc
tatccatgcc aaacgtgtaa caattatgcc 480aaaagacatc cagctagcac
gccgcatacg tggagaacgt gcttaagaat ccactatgat 540gggaaacatt
tcattctcaa aaaaaaaaaa aaaaatttct cttcttcctg ttattggtag
600ttctgaacgt tagatatttt ttttccatgg ggtcaaaagg tacctaagta
tatgattgcg 660agtggaaaaa taggggacag aaatcaggta ttggcagttt
ttccattttc atttgtgtgt 720gaatttttaa tataaatgcg gagacgtaaa
gcattaatgc aagttaaaat gtttcagtga 780acaagtttca gcggttcaac
tttataataa ttataaataa acctgttaaa tttttctgga 840caatgccagc
atttggattt ttttaaaaca agtaaatttc ttattgatgg caactaaatg
900gtgtttgtag catttttatc atacagtaga ttccatccat tcactatact
tttctaactg 960agttgtccta catgcaagta catgttttta atgttgtctg
tcttctgtgc tgttcctgta 1020agtttgctat taaaatacat taaactataa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaa 1117641665DNAHomo sapiensmisc_featureHomo
sapiens interleukin 8 (IL8), mRNA NM_000584 64ctccataagg cacaaacttt
cagagacagc agagcacaca agcttctagg acaagagcca 60ggaagaaacc accggaagga
accatctcac tgtgtgtaaa catgacttcc aagctggccg 120tggctctctt
ggcagccttc ctgatttctg cagctctgtg tgaaggtgca gttttgccaa
180ggagtgctaa agaacttaga tgtcagtgca taaagacata ctccaaacct
ttccacccca 240aatttatcaa agaactgaga gtgattgaga gtggaccaca
ctgcgccaac acagaaatta 300ttgtaaagct ttctgatgga agagagctct
gtctggaccc caaggaaaac tgggtgcaga 360gggttgtgga gaagtttttg
aagagggctg agaattcata aaaaaattca ttctctgtgg 420tatccaagaa
tcagtgaaga tgccagtgaa acttcaagca aatctacttc aacacttcat
480gtattgtgtg ggtctgttgt agggttgcca gatgcaatac aagattcctg
gttaaatttg 540aatttcagta aacaatgaat agtttttcat tgtaccatga
aatatccaga acatacttat 600atgtaaagta ttatttattt gaatctacaa
aaaacaacaa ataattttta aatataagga 660ttttcctaga tattgcacgg
gagaatatac aaatagcaaa attgaggcca agggccaaga 720gaatatccga
actttaattt caggaattga atgggtttgc tagaatgtga tatttgaagc
780atcacataaa aatgatggga caataaattt tgccataaag tcaaatttag
ctggaaatcc 840tggatttttt tctgttaaat ctggcaaccc tagtctgcta
gccaggatcc acaagtcctt 900gttccactgt gccttggttt ctcctttatt
tctaagtgga aaaagtatta gccaccatct 960tacctcacag tgatgttgtg
aggacatgtg gaagcacttt aagttttttc atcataacat 1020aaattatttt
caagtgtaac ttattaacct atttattatt tatgtattta tttaagcatc
1080aaatatttgt gcaagaattt ggaaaaatag aagatgaatc attgattgaa
tagttataaa 1140gatgttatag taaatttatt ttattttaga tattaaatga
tgttttatta gataaatttc 1200aatcagggtt tttagattaa acaaacaaac
aattgggtac ccagttaaat tttcatttca 1260gataaacaac aaataatttt
ttagtataag tacattattg tttatctgaa attttaattg 1320aactaacaat
cctagtttga tactcccagt cttgtcattg ccagctgtgt tggtagtgct
1380gtgttgaatt acggaataat gagttagaac tattaaaaca gccaaaactc
cacagtcaat 1440attagtaatt tcttgctggt tgaaacttgt ttattatgta
caaatagatt cttataatat 1500tatttaaatg actgcatttt taaatacaag
gctttatatt tttaacttta agatgttttt 1560atgtgctctc caaatttttt
ttactgtttc tgattgtatg gaaatataaa agtaaatatg 1620aaacatttaa
aatataattt gttgtcaaag taaaaaaaaa aaaaa 166565366DNAPrevotella
melaninogenicamisc_featurePrevotella melaninogenica partial 16S
rRNA gene NCBI AJ555137 65ggctcaggat gattttctag ctacaggctt
aacacatgca agtcgagggg aaacggcatt 60gagtgcttgc actctttgga cgtcgaccgg
cgcacgggtg agtaacgcgt atccaacctt 120cccattactg tgggataacc
tgccgaaagg cagactaata cctgcatagt cttcgatgac 180ggcatcagat
ttgaagtaaa gatttatcgg taatggatgg ggatgcgtct gattagcttg
240ttggcggggt aacggcccac caaggctacg atcagtaggg gttctgagag
gaaggtcccc 300cacattggaa ctgagacacg gcccaaactc ctacgggagg
cagcaatcga attcccgctt 360gccaca 366661454DNAStreptococcus
mitismisc_featureStreptococcus mitis partial 16S rRNA gene, strain
B6 NCBI AJ617805 66cgctggcggc gtgcctaata catgcaagta gaacgctgaa
ggaggagctt gcttctctgg 60atgagttgcg aacgggtgag taacgcgtag gtaacctgcc
tggtagcggg ggataactat 120tggaaacgat agctaatacc gcataagagt
agatgttgca tgacatttgc ttaaaaggtg 180caattgcatc actaccagat
ggacctgcgt tgtattagct agttggtggg gtaacggctc 240accaaggcga
cgatacatag ccgacctgag agggtgatcg gccacactgg gactgagaca
300cggcccagac tcctacggga ggcagcagta gggaatcttc ggcaatggac
ggaagtctga 360ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt
aaagctctgt tgtaagagaa 420gaacgagtgt gagagtggaa agttcacact
gtgacggtat cttaccagaa agggacggct 480aactacgtgc cagcagccgc
ggtaatacgt aggtcccgag cgttgtccgg atttattggg 540cgtaaagcga
gcgcaggcgg ttagataagt ctgaagttaa aggctgtggc ttaaccatag
600tacgctttgg aaactgttta acttgagtgc aagaggggag agtggaattc
catgtgtagc 660ggtgaaatgc gtagatatat ggaggaacac cggtggcgaa
agcggctctc tggcttgtaa 720ctgacgctga ggctcgaaag cgtggggagc
aaacaggatt agataccctg gtagtccacg 780ccgtaaacga tgagtgctag
gtgttagacc ctttccgggg tttagtgccg cagctaacgc 840attaagcact
ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg
900gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa
ccttaccagg 960tcttgacatc cctctgaccg ctctagagat agagttttcc
ttcgggacag aggtgacagg 1020tggtgcatgg ttgtcgtcag ctcgtgtcgt
gagatgttgg gttaagtccc gcaacgagcg 1080caacccctat tgttagttgc
catcatttag ttgggcactc tagcgagact gccggtaata 1140aaccggagga
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca
1200cgtgctacaa tggctggtac aacgagtcgc aagccggtga cggcaagcta
atctcttaaa 1260gccagtctca gttcggattg taggctgcaa ctcgcctaca
tgaagtcgga atcgctagta 1320atcgcggatc agcacgccgc ggtgaatacg
ttcccgggcc ttgtacacac cgcccgtcac 1380accacgagag tttgtaacac
ccgaagtcgg tgaggtaacc gtaaggagcc agccgcctaa 1440ggtttgatag atga
1454671480DNACapnocytophaga gingivalismisc_featureCapnocytophaga
gingivalis 16S ribosomal RNA gene NCBI AF543295 67agagtttgat
cctggctcag atgaacgcta gcggcaggcc taacacatgc aagtcgaggg 60agaagccctt
tcgggggcag aaaccggcgc acgggtgcgt aacgcgtatg caacctacct
120ttcacagggg gatagcccga agaaatttgg attaataccc cataatatta
ttggatggca 180tcatttgata attaaaactg cggtggtgaa agatgggcat
gcgtcctatt agctagttgg 240agtggtaacg gcaccccaag gctacgatag
gtaggggtcc tgagagggag atcccccaca 300ctggtactga gacagggacc
agacccctac gggaggcagc agtgaggaat attggtcaat 360ggtcggaaga
ctgaaccagc catgccgcgt gcaggaagaa tgccttatgg gttgtaaact
420gcttttatat gggaagaata aggtgtacgt gtacattgat gacggtacca
tatgaataag 480catcggctaa ctccgtgcca gcagccgcgg taatacggag
gatgcgagcg ttattcggaa 540tcattgggtt taaagggtct gtaggcgggc
tattaagtca ggggtgaaag gtttcagctt 600aactgagaaa ttgcctttga
tactggtagt cttgaatatc tgtgaagttc ttggaatgtg 660tagtgtagcg
gtgaaatgct tagatattac acagaacacc gattgcgaag gcaggggact
720aacagacaat tgacgctgag agacgaaagc gtggggagcg aacaggaatt
agatcccctg 780gtagtccacg cctgtaaacg atggatacta gctgttgggc
gcaggctgag ttggcttaag 840cgaaagtgat aagtatccaa ccttggggaa
gtacgcacgc
aagtgtgaaa ctcaaaggaa 900ttgacggggg cccgcacaag cggtggagca
tgtggtttaa ttcgatgata cgcgaggaac 960cttaccaagg tttaaatgga
gactgacagg tgtagagata cgcccttctt cggacagttt 1020tcaaggtgct
gcatggttgt cgtcagctcg tgccgtgagg tgtcaggtta agtcctataa
1080cgagcgcaac ccctattgtt agttaccagc aagtaaagtt ggggactcta
gcaagactgc 1140cggtgtaaac cgtgaggaag gtggggatga cgtcaaatca
tcacggccct tacatcttgg 1200gctacacacg tgctacaatg gtcgttacag
agagcagcca ctgcgcgagc aggagcgaat 1260ctataaagac ggtcacagtt
cggatcggag tctgcaactc gactccgtga agctggaatc 1320gctagtaatc
ggatatcagc catgatccgg tgaatacgtt cccgggcctt gtacacaccg
1380cccgtcaagc catggaagct gggagtacct gaagtcggtc accgcaagga
gctgcctagg 1440gtaaaaccag tgactggggc taagtcgtaa caaggtaacc
148068838DNAMicrococcus luteusmisc_featureMicrococcus luteus
partial 16S rRNA gene, isolate A24 NCBI AM285006 68aggatgaacg
ctggcggcgt gcttacacat gcaagtcgaa cgatgaagcc cagcttgctg 60ggtggattag
tggcgaacgg gtgagtaaca cgtgagtaac ctgcccttaa ctctgggata
120agcctgggaa actgggtcta ataccggata ggagcgtcca ccgcatggtg
ggtgttggaa 180agatttatcg gttttggatg gactcgcggc ctatcagctt
gttggtgagg taatggctca 240ccaaggcgac gacgggtagc cggcctgaga
gggtgaccgg ccacactggg actgagacac 300ggcccagact cctacgggag
gcagcagtgg ggaatattgc gcaatgggcg caagcctgat 360gcagcgacgc
cgcgtgaggg atgacggcct tcgggttgta aacctctttc agtagggaag
420aagcgaaagt gacggtacct gcagaagaag caccggctaa ctacgtgcca
gcagccgcgg 480taatacgtag ggtgcgagcg ttatccggaa ttattgggcg
taaagagctc gtaggcggtt 540tgtcgcgtct gtcgtgaaag tccggggctt
aaccccggat ctgcggtggg tacgggcaga 600ctagagtgca gtaggggaga
ctggaattcc tggtgtagcg gtggaatgcg cagatatcag 660gaggaacacc
gatggcgaag gcaggtctct gggctgtaac tgacgctgag gagcgaaagc
720atggggagcg aacaggatta gataccctgg tagtccatgc cgtaaacgtt
gggcactagg 780tgtggggacc attccacggt tttccgcgcc gcagctaacg
cattaagtgc cccgccct 83869297DNAHuman papillomavirus type
16misc_featureHuman papillomavirus type 16 strain P209 E7 protein
(E7) gene, complete cds NCBI EF422141 69atgcatggag atacacctac
attgcatgaa tatatgttag atttgcaacc agagacaact 60gatctctact gttatgagca
attaaatgac agctcagagg aggaggatga aatagatggt 120ccagctggac
aagcagaacc ggacagagcc cattacaata ttgtaacctt ttgttgcaag
180tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg
tactttggaa 240gacctgttaa tgggcacact aggaattgtg tgccccatct
gttctcagaa accataa 29770477DNAHuman papillomavirus type
18misc_featureHuman papillomavirus type 18 strain P629 E6 protein
(E6) gene, complete cds NCBI EF422111 70atggcgcgct ttgaggatcc
aacacggcga ccctacaagc tacctgatct gtgcacggaa 60ctgaacactt cactgcaaga
catagaaata acctgtgtat attgcaagac agtattggaa 120cttacagagg
tatttgaatt tgcattcaaa gatttatttg tagtgtatag agacagtata
180ccgcatgctg catgccataa atgtatagat ttttattcta gaattagaga
attaagacat 240tattcagact ctgtgtatgg agacacatta gaaaaactaa
ctaacactgg gttatacaat 300ttattaataa ggtgcctgcg gtgccagaaa
ccgttgaatc cagcagaaaa acttagacac 360cttaatgaaa aacgacgatt
ccacaaaata gctgggcact atagaggcca gtgccattcg 420tgctgcaacc
gagcacgaca ggagagactc caacgacgca gagaaacaca agtataa 477
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